Modulators of myc, methods of using the same, and methods of identifying agents that modulate myc

ABSTRACT

Disclosed herein are methods of modulation of the viability of a cell. Further disclosed herein are methods of modulating an immune response. Further disclosed herein are methods of identifying agents capable of modulation of the viability of a cell or an immune response. Further disclosed herein are agents and compositions capable of modulation of the viability of a cell or an immune response.

CROSS-REFERENCE

This application is a Continuation application of U.S. application Ser.No. 12/550,166, with a filing date of Aug. 28, 2009, which claims thebenefit of U.S. Provisional Application No. 61/092,708, filed Aug. 28,2008, all of which are incorporated herein by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 691772000501SEQLIST.txt,date recorded: Feb. 26, 2013, size: 9 KB).

BACKGROUND OF THE INVENTION

There are multiple types of vaccines: vaccines containing deadmicroorganisms (e.g. the influenza and cholera vaccines); vaccinescontaining attenuated microorganisms (e.g. the MMR vaccine); vaccinescontaining toxoids (e.g. the DPT vaccine); vaccines containing subunitsof the pathogen (e.g. the HPV vaccine); and nucleic acid (e.g. RNA andDNA) vaccines (e.g. avian flu vaccine, West Nile Virus vaccine, andmultiple cancer vaccines). Vaccine adjuvants are agents that stimulatethe immune system and increase the response of the immune system to avaccine.

SUMMARY OF THE INVENTION

There is a need for vaccine adjuvants that enhance an immunologicalresponse to an antigen. The inventors have discovered that increasingthe expression of MYC or the activity of a MYC peptide enhancesimmunological responses to antigens. In order to increase MYCconcentrations in the nucleus of cells with minimal side-effects, theinventors have designed a fusion peptide that can penetrate the nucleusof cells. This peptide can be administered topically to decreasesystemic effects. This peptide also increases the viability ofleukocytes, including T-cells, and B-cells. Further, the inventors havedesigned several assays for identifying other agents that meet thisunmet need.

There is also a need for methods of treating autoimmune disorders. Theinventors have discovered that decreasing the expression of MYC or theactivity of a MYC peptide treats auto-immune disorders. In order tofulfill this unmet need, the inventors have designed several assays foridentifying agents that inhibit (partially or fully) the activity of animmune system. In certain instances, the agents that inhibit the immunesystem decrease the viability of leukocytes or increase the proportionof anergic B-cells.

Disclosed hereon, in certain embodiments, is a peptide that up-regulatesthe expression of a MYC gene, the activity of a Myc polypeptide, or acombination thereof, comprising: (a) a transporter peptide sequence; (b)a MYC sequence; and optionally (c) one or more molecules that link thetransporter peptide sequence and the MYC sequence. In some embodiments,the peptide has Formula (I):

-   -   transporter peptide sequence —MYC sequence.        In some embodiments, the peptide has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein —X— is a molecule that links the transporter peptide        sequence and the MYC sequence. In some embodiments, the peptide        has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein in X is at least one amino acid. In some embodiments,        the peptide has the following amino acid sequence:

MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.

Disclosed herein, in certain embodiments, is a composition that inducesan immune response, comprising a fusion peptide comprising: (a) atransporter peptide sequence; (b) a MYC sequence; and optionally (c) oneor more molecules that link the transporter peptide sequence and the MYCsequence. In some embodiments, the peptide has Formula (I):

-   -   transporter peptide sequence —MYC sequence.        In some embodiments, the peptide has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein —X— is a molecule that links the transporter peptide        sequence and the MYC sequence. In some embodiments, the peptide        has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein in X is at least one amino acid. In some embodiments,        the peptide has the following amino acid sequence:

MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.In some embodiments, the composition further comprises an antigen,antigenic moiety, or a combination thereof. In some embodiments, theantigen is a dead microorganism, an attenuated microorganism, a toxoid,a subunit of a pathogen, a nucleic acid, a polymer of nucleic acids, orcombinations thereof. In some embodiments, the antigen is derived from:hepatitis A; hepatitis B; polio; measles; mumps; rubella; diphtheria;pertussis; tetanus; influenza; varicella zoster virus; rotavirus;meningococcal; pneumonia; smallpox; cholera; bubonic plague; yellowfever; tuberculosis; human papillomavirus; or combinations thereof. Insome embodiments, the composition further comprises an antigenic moietyderived from a pathogen selected from: hepatitis A; hepatitis B; polio;measles; mumps; rubella; diphtheria; pertussis; tetanus; influenza;varicella zoster virus; rotavirus; meningococcal; pneumonia; smallpox;cholera; bubonic plague; yellow fever; tuberculosis; humanpapillomavirus; or combinations thereof. In some embodiments, thecomposition further comprises an antigenic moiety derived from aneoplastic cell. In some embodiments, the composition is formulated fortopical administration.

Disclosed herein, in certain embodiments, is a method of inducing animmune response, comprising administering to an individual in needthereof: (a) a vaccine against at least one antigen, and (b) a fusionpeptide comprising: (a) a transporter peptide sequence; (b) a MYCsequence; and optionally (c) one or more molecules that link thetransporter peptide sequence and the MYC sequence. In some embodiments,the peptide has Formula (I):

-   -   transporter peptide sequence —MYC sequence.        In some embodiments, the peptide has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein —X— is a molecule that links the transporter peptide        sequence and the MYC sequence. In some embodiments, the peptide        has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein in X is at least one amino acid. In some embodiments,        the peptide has the following amino acid sequence:

MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.In some embodiments, the vaccine is selected from the group consistingof: hepatitis A vaccine; hepatitis B vaccine; polio vaccine; measlesvaccine; mumps vaccine; rubella vaccine; diphtheria vaccine; pertussisvaccine; tetanus vaccine; influenza vaccine; varicella zoster virusvaccine; rotavirus vaccine; meningococcal vaccine; pneumonia vaccine;smallpox vaccine; cholera vaccine; bubonic plague vaccine; yellow fevervaccine; tuberculosis vaccine; human paplomavirus vaccine; a cancervaccine; or combinations thereof. In some embodiments, the vaccine isadministered before, after, or simultaneously with the agent thatincreases the nucleic concentration of a Myc polypeptide.

Disclosed herein, in certain embodiments, is a method of increasing theviability of a cell, comprising administering to an individual in needthereof an agent that up-regulates the expression of MYC, increases theactivity of a Myc peptide, or a combination thereof. In someembodiments, the agent is a fusion peptide comprising: (a) a transporterpeptide sequence; (b) a MYC sequence; and optionally (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence. In some embodiments, the peptide has Formula (I):

-   -   transporter peptide sequence —MYC sequence.        In some embodiments, the peptide has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein —X— is a molecule that links the transporter peptide        sequence and the MYC sequence. In some embodiments, the peptide        has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein in X is at least one amino acid. In some embodiments,        the peptide has the following amino acid sequence:

MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHHIn some embodiments, the cell is a leukocyte. In some embodiments, thecell is a T-cell. In some embodiments, the cell is a B-cell. In someembodiments, the cell is a memory T-cell.

Disclosed herein, in certain embodiments, is a method of decreasing theviability of a cell, comprising administering to an individual in needthereof an agent that down-regulates the expression of MYC, decreasesthe activity of a Myc peptide, or a combination thereof.

Disclosed herein, in certain embodiments, is a method of treating adisorder characterized by the under-expression of a MYC gene or adeficit in the activity of a Myc polypeptide, comprising administeringto an individual in need thereof an agent that up-regulates theexpression of MYC, increases the activity of a Myc peptide, or acombination thereof. In some embodiments, the agent is a fusion peptidecomprising: (a) a transporter peptide sequence; (b) a MYC sequence; andoptionally (c) one or more molecules that link the transporter peptidesequence and the MYC sequence. In some embodiments, the peptide hasFormula (I):

-   -   transporter peptide sequence —MYC sequence.        In some embodiments, the peptide has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein —X— is a molecule that links the transporter peptide        sequence and the MYC sequence. In some embodiments, the peptide        has Formula (II):    -   transporter peptide sequence—X—MYC sequence,        wherein in X is at least one amino acid. In some embodiments,        the peptide has the following amino acid sequence:

MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.

Disclosed herein, in certain embodiments, is a method of treating adisorder characterized by the over-expression of a MYC gene or theexcess activity of a Myc polypeptide comprising, administering to anindividual in need thereof an agent that down-regulates the expressionof MYC, decreases the activity of a Myc peptide, or a combinationthereof.

Disclosed herein, in certain embodiments, is a method of identifying anagent that up-regulates the expression of a MYC gene, and/or theactivity of a Myc polypeptide, the method comprising: (a) contacting aplurality of anergic B-cells with an agent; and (b) following contactwith the agent, detecting and/or measuring the level of expression ofone or more cell surface markers in the cell culture, wherein thepresence of the cell surface marker is indicative a non-anergic B-cell.In some embodiments, the anergic B-cells are obtained from mice with thephenotype BCR^(HEL)/sHEL. In some embodiments, the cell surface markeris: IgM, IgMa, IgMb, B220, CD21/35, CD23, CD24 (HSA), CD40, CD69, CD80and/or CD86 (B7-2). In some embodiments, the level of expression of acell surface marker is measured by contacting the plurality of cellswith a detectable antibody or detectable antigen that binds to the cellsurface marker.

Disclosed herein, in certain embodiments, is a method of identifying anagent that up-regulates the expression of a MYC gene, and/or theactivity of a Myc polypeptide, the method comprising: (a) contacting aplurality of factor-dependent cells with an agent; and (b) followingcontact with the agent, detecting and measuring the level of expansionof the plurality of cells. In some embodiments, the factor-dependentcells are lymphoid cells. In some embodiments, the factor-dependentcells are: IL-2^(-/-), IL-3^(-/-), IL-4^(-/-), IL-5^(-/-), IL-6^(-/-),IL-7^(-/-), IL-8^(-/-), IL-9^(-/-), IL-10^(-/-), IL-11^(-/-),IL-12^(-/-), or any combinations thereof. In some embodiments, thefactor-dependent cells are derived from: CTLL-2 cells or BAF/3 cells.

Disclosed herein, in certain embodiments, is a method of identifying anagent that up-regulates the expression of a MYC gene, and/or theactivity of a Myc polypeptide, comprising: (a) transforming a pluralityof cells with a reporter construct, comprising: a reporter gene operablylinked to an E-box sequence encoded in a myc-responsive promoter; (b)contacting the plurality of cells with an agent; and (c) followingcontact with the agent, detecting and measuring the level of expressionof the reporter gene. In some embodiments, the cells are lymphoid cells.In some embodiments, the myc-responsive promoter is an ornithinedecarboxylase promoter. In some embodiments, the reporter gene is aβ-galactosidase gene, a β-lactamase gene, a horseradish peroxidase gene,an alkaline phosphatase gene, a thymidine kinase gene, a xanthinephosphoribotransferase gene, a tyrosinase gene, a cytosine deaminasegene, an antibiotic resistance gene, or a gene having a fluorescentexpression product.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein, in certain embodiments, are methods of identifyingagents capable of modulating (e.g., activating, increasing, inducing,supplementing, decreasing, or inhibiting) an immune system. In someembodiments, agents are utilized to increase an immune response. In someembodiments, agents are used to decrease an immune response. Further,disclosed herein are methods of modulating an immune system using agentsidentified by any of the means disclosed herein.

Certain Definitions

Unless indicated otherwise, the following terms have the followingmeanings when used herein and in the appended claims.

The term “vaccine” means a composition comprising antigens and/orantigenic moieties administered to a mammal in order to elicit an immuneresponse. The antigen or antigenic moiety is a live or attenuatedmicroorganism, a natural product purified from a microorganism (e.g. asubunit of a protein, peptide, polysaccharide, and a nucleic acid), asynthetic product designed to mimic a component of the microorganism, agenetically engineered protein, peptide, or polysaccharide, a patient'sown tumor cells, a natural product purified from a tumor cell (e.g.Prostate Specific Antigen, gp96, GM2, GD2, GD3, carcinoembryonicantigen, MART-1, and tyrosinase), a synthetic molecule designed to mimica component of a tumor cell (e.g. sialyl Tn), or combinations thereofFurther, the term vaccine includes current vaccines, and any noveland/or modified vaccines developed.

The term “lymphoid tissue” means tissue associated with the lymphaticsystem. By way of non-limiting example, lymphoid tissue includes tissueobtained from the lymph nodes, tonsils, spleen, bone marrow, and thymus.

The term “lymphocyte” refers to all immature, mature, undifferentiatedand differentiated white lymphocyte populations including tissuespecific and specialized varieties. It encompasses, by way ofnon-limiting example, B-cells, T-cells, NKT cells, and NK cells. In someembodiments, lymphocytes include all B-cell lineages includingpre-B-cells, Progenitor B cells, Early Pro-B cells, Late Pro-B cells,Large Pre-B cells, Small Pre-B cells, Immature B cells, Mature B cells,plasma B-cells, memory B-cells, B-1 cells, B-2 cells and anergic AN1/T3cell populations.

The term B-cell, refers to, by way of non-limiting example, apre-B-cell, Progenitor B cell, Early Pro-B cell, Late Pro-B cell, LargePre-B cell, Small Pre-B cell, Immature B cell, Mature B cell, plasmaB-cell, memory B-cell, B-1 cell, B-2 cells and anergic AN1/T3 cellpopulations. In some embodiments, the term B-cell includes a B-cell thatexpresses an immunoglobulin heavy chain and/or light chain on its cellssurface. In some embodiments, the term B-cell includes a B-cell thatexpresses and secretes an immunoglobulin heavy chain and/or light chain.In some embodiments, the term B-cell includes a cell that binds anantigen on its cell-surface. In some embodiments disclosed herein,B-cells or AN1/T3 cells are utilized in the processes described. Incertain embodiments, such cells are optionally substituted with anyanimal cell suitable for expressing, capable of expressing (e.g.,inducible expression), or capable of being differentiated into a cellsuitable for expressing an antibody including, e.g., a hematopoieticstem cell, a B-cell, a pre-B-cell, a Progenitor B cell, a Early Pro-Bcell, a Late Pro-B cell, a Large Pre-B cell, a Small Pre-B cell, anImmature B cell, a Mature B cell, a plasma B-cell, a memory B-cell, aB-1 cell, a B-2 cell, an anergic B-cell, or an anergic AN1/T3 cell.

The term “immune response” includes the identification andneutralization of pathogens and/or tumor cells. In some embodiments, theimmune response is the adaptive immune response. By way of non-limitingexample, the adaptive immune response includes the development ofimmunological memory.

The terms “antibody” and “antibodies” refer to any form of a naturaloccurring (but isolated and/or purified), engineered or syntheticantibody, including monoclonal antibodies, polyclonal antibodies,bi-specific antibodies, multispecific antibodies, grafted antibodies,human antibodies, humanized antibodies, synthetic antibodies, chimericantibodies, camelized antibodies, single-chain Fvs (scFv), single chainantibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs(sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies andantigen-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ andIgA₂) or subclass. The terms “antibody” and immunoglobulin are usedinterchangeably in the broadest sense. In some embodiments an antibodyis part of a larger molecule, formed by covalent or non-covalentassociation of the antibody with one or more other proteins or peptides.

The antibodies herein include monoclonal, polyclonal, recombinant,chimeric, humanized, bi-specific, grafted, human, and fragments thereofincluding antibodies altered by any means to be less immunogenic inhumans. Thus, for example, the monoclonal antibodies and fragments,etc., herein include “chimeric” antibodies and “humanized” antibodies.In general, chimeric antibodies include a portion of the heavy and/orlight chain that is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, so long as they exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567); Morrison et al. Proc. Natl Acad.Sci. 81:6851-6855 (1984). For example, in some embodiments a chimericantibody contains variable regions derived from a mouse and constantregions derived from human in whom the constant region containssequences homologous to both human IgG2 and human IgG4. “Humanized”forms of non-human (e.g., murine) antibodies or fragments are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include, grafted antibodies or CDR graftedantibodies wherein part or all of the amino acid sequence of one or morecomplementarily determining regions (CDRs) derived from a non-humananimal antibody is grafted to an appropriate position of a humanantibody while maintaining the desired binding specificity and/oraffinity of the original non-human antibody. In some embodiments,corresponding non-human residues replace Fv framework residues of thehuman immunoglobulin. In some embodiments humanized antibodies compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences. These modifications are made tofurther refine and optimize antibody performance. In some embodiments,the humanized antibody comprises substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. For further details, see, e.g.: Joneset al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332:323-329 (1988) and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

The term “antigen” refers to a substance that elicits the production ofan antibody.

The term “antigenic moiety” means the portion of a molecule, organism(e.g., a bacteria, virus), or cell to which an antibody binds (orcomplements).

The term “self antigen” refers to an antigen that originates from withinan animal, tissue, or cell. In some embodiments a self antigen comprisesan endogenous antigen. In some embodiments a self antigen comprises anendogenous antigen produced by an endogenous retrovirus. In someembodiments self antigens comprise neo-self antigens, microbially orparasite encoded neo-self antigens, or other neo-self antigens expressedas a result of genetic alteration to an animal or cell followingwhole-body irradiation and HSC transplantation (which re-starts theimmune system and allows the new antigen to be considered “self”). Insome embodiments a chimeric mouse expresses a neo-self antigen.

The term “auto-antigen” refers to an antigen that comprises an epitopeof a self antigen or an immunologically reactive epitope that mimicsthat of a self antigen. In some embodiments the term auto antigencomprises antigens to which autoantibodies are produced. In someembodiments an auto antigen comprises an endogenous antigen wherein theanimal from which the endogenous antigen originated is or was onceimmunologically tolerant to the antigen.

The term “anergy” refers to a state wherein lymphocytes are inactive(e.g. they do not respond to the binding of an antigen). In someembodiments, the lymphocyte is a B-cell. In some embodiments, a B-cellbecomes anergic following exposure to a circulating solubleself-antigen. In some embodiments, a B-cell becomes anergic followingexposure to a soluble circulating antigen. In some embodiments, theantigen is soluble hen egg lysozyme (sHEL).

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to agents that have the same basic chemical structure asa naturally occurring amino acid, i.e., an a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The terms “polypeptide”, peptide” and “protein” are used interchangeablyherein to refer to a polymer of amino acid residues. The terms apply tonaturally occurring amino acid polymers as well as amino acid polymersin which one or more amino acid residues is a non-naturally occurringamino acid, e.g., an amino acid analog. The terms encompass amino acidchains of any length, including full length proteins, wherein the aminoacid residues are linked by covalent peptide bonds.

The terms “Myc protein” and “Myc polypeptide” are used synonyms andrefer to the polymer of amino acid residues disclosed in NCBI AccessionNumber NP002458.2, and functional homologs, analogs or fragmentsthereof. In some embodiments, a Myc polypeptide comprises an amino acidsequence that is at least 40% to 100% identical, e.g., at least 40%,45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%,92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 40% toabout 100% identical to the sequence of NCBI Accession NumbersNP002458.2. In some embodiments, a Myc polypeptide comprises apolypeptide sequence of 40 amino acids or more in length that is atleast 50% to 100% identical, e.g., at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, orany other percent from about 50% to about 100% identical to the sequenceof NCBI Accession Numbers NP002458.2. In some embodiments, a Mycpolypeptide comprises a polypeptide sequence of 40 amino acids or morein length that is at least 50% to 100% identical, e.g., at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%,95%, 96%, 97%, 98%, or any other percent from about 50% to about 100%identical to the sequence of NCBI Accession Numbers NP002458.2. In someembodiments, Myc polypeptide means a polymer of 439 amino acids, a Mycpolypeptide that has not undergone any post-translational modifications,and/or a Myc polypeptide that has undergone post-translationalmodifications. In certain instances, the Myc polypeptide is 48,804 kDa.In certain instances, the Myc polypeptide contains a basicHelix-Loop-Helix Leucine Zipper (bHLH/LZ) domain. In certain instances,the Myc polypeptide is a transcription factor (e.g. Transcription Factor64). In certain instances, the Myc polypeptide binds to a sequencecomprising CACGTG (i.e. an E-box sequence).

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions are achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “MYC” and “MYC gene” are synonyms. They refer to a nucleicacid sequence that encodes a Myc polypeptide. A MYC gene comprises anucleotide sequence of at least 120 nucleotides that is at least 60% to100% identical or homologous, e.g., at least 60, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 70% to about 100% identical to sequences of NCBIAccession Number NM 002467. In some embodiments, the MYC gene is aproto-oncogene. In certain instances, a MYC gene is found on chromosome8, at 8q24.21. In certain instances, a MYC gene begins at 128,816,862 bpfrom pter and ends at 128,822,856 bp from pter. In certain instances, aMYC gene is about 6 kb. In certain instances, a MYC gene encodes atleast eight separate mRNA sequences—5 alternatively spliced variants and3 unspliced variants.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences can be aligned for optimal comparisonpurposes (e.g., gaps are introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions can then becompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments the two sequences are the samelength.

To determine percent homology between two sequences, the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877 is used. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.215:403-410. BLAST nucleotide searches are performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described or disclose herein.BLAST protein searches are performed with the XBLAST program, score=50,wordlength=3. To obtain gapped alignments for comparison purposes,Gapped BLAST is utilized as described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) are used. See the website of the National Center forBiotechnology Information for further details (on the World Wide Web atncbi.nlm.nih.gov). Proteins suitable for use in the methods describedherein also includes proteins having between 1 to 15 amino acid changes,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acidsubstitutions, deletions, or additions, compared to the amino acidsequence of any protein described herein. In other embodiments, thealtered amino acid sequence is at least 75% identical, e.g., 77%, 80%,82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of any protein inhibitor described herein. Suchsequence-variant proteins are suitable for the methods described hereinas long as the altered amino acid sequence retains sufficient biologicalactivity to be functional in the compositions and methods describedherein. Where amino acid substitutions are made, the substitutionsshould be conservative amino acid substitutions. Among the common aminoacids, for example, a “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine. The BLOSUM62 table is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff et al (1992), Proc.Natl Acad. Sci. USA, 89:10915-10919). Accordingly, the BLOSUM62substitution frequencies are used to define conservative amino acidsubstitutions that, in some embodiments, are introduced into the aminoacid sequences described or disclosed herein. Although it is possible todesign amino acid substitutions based solely upon chemical properties(as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

The term “Myc activity” refers to binding of a Myc polypeptide to anucleic acid sequence. In some embodiment, MYC activity further includesMYC regulation of the transcriptional activity of Myc responsive genes.In some embodiments, Myc activity induces cell proliferation and/orantibody production.

The terms “activation of Myc” and “Myc activation” refer to theinduction of Myc activity. In some embodiments activation of Myc isinduced by over-expression of a Myc polypeptide. In some embodimentsactivation of Myc is induced by transport of a Myc polypeptide into thenucleus of a cell. In some embodiments activation of Myc is induced bytransport of a Myc polypeptide into a cell.

The term “expression” refers to one or more of the following events: (1)production of an RNA template from a DNA sequence (e.g., bytranscription) within a cell; (2) processing of an RNA transcript (e.g.,by splicing, editing, 5′ cap formation, and/or 3′ end formation) withina cell; (3) translation of an RNA sequence into a polypeptide or proteinwithin a cell; (4) post-translational modification of a polypeptide orprotein within a cell; (5) presentation of a polypeptide or protein onthe cell surface; (6) secretion or release of a polypeptide or proteinfrom a cell.

The term “endogenous” in the context of a cellular protein refers toprotein naturally occurring and/or expressed by the cell in the absenceof recombinant manipulation; accordingly, the terms “endogenouslyexpressed protein” or “endogenous protein” excludes cellular proteinsexpressed by means of recombinant technology.

The term “factor-dependent cell” means a cell that is not able tosurvive and/or proliferate without contact with an exogenous growthfactor (e.g. a cytokine). When contacted with the requiredgrowth-factors, a factor-dependent cell is able to survive and/orproliferate. In the absence of the required growth-factors, afactor-dependent cell cannot survive (e.g. will undergo apoptosis)and/or proliferate.

The term “expansion” as used herein refers to an enlargement in the sizeof the culture due to the proliferation (i.e. division) of cells in thecell culture. In some embodiments, the cell culture exhibits expansion(i.e. it attains a larger size and/or the concentration of cell in theculture increases). In some embodiments, the cell culture exhibitsnegative expansion (i.e. attains a smaller size or the concentration ofcell in the culture decreases). In certain instances, the expansion of acell culture is detectable and measurable in any suitable manner (e.g.staining, flow cytometry, fluorescent microscopy, HPLC, confocalmicroscopy, infrared spectroscopy, autoradiography).

The phrases “E-box sequence” and “enhancer box sequence” are usedinterchangeably herein and mean the nucleotide sequence CANNTG, whereinN is any nucleotide. In certain instances, the E-box sequence comprisesCACGTG. In certain instances, the basic helix-loop-helix domain of atranscription factor encoded by MYC binds to the E-box sequence. Incertain instances the E-box sequence is located upstream of a gene (e.g.p21, Bc1-2, or ornithine decarboxylase). In certain instances, thebinding of the transcription factor encoded by MYC to the E-boxsequence, allows RNA polymerase to transcribe the gene downstream of theE-box sequence.

Methods of Modulating the Viability of a Cell

Disclosed herein, in some embodiments, are methods of modulating theviability of a cell. As used herein, “viability” means the length oftime the cell survives, the rate (or amount) of cellular proliferation,or a combination thereof. In some embodiments, the viability of a cellis increased. In some embodiments, the viability of a cell is decreased.

In some embodiments, the modulation occurs in vivo. In some embodiments,the modulation occurs in vitro. In some embodiments, a method ofmodulating the viability of a cell comprises modulating the viability ofa leukocyte. In some embodiments, the leukocyte is a T-cell. In someembodiments, the leukocyte is a CD4+ T-cell. In some embodiments, theleukocyte is a memory T-cell.

In some embodiments, modulating the viability of a cell comprisesmodulating the expression of a MYC gene, and/or the activity of a Mycpolypeptide. In some embodiments, modulating the expression of a MYCgene, and/or the activity of a Myc polypeptide directly or indirectlyresults in the modulation of a gene or polypeptide regulated by MYC. Insome embodiments, the gene or polypeptide directly or indirectlyregulated by a MYC transcription factor is: neurogranin, MEF-2c, gfi-1,cyclin D2, CDK4, Egr-2, Nab-2, TGIF, NF-kB p105, Carma-1, A1 or Bc1-2.

Increasing the Viability of a Cell

In some embodiments, modulating the viability of a cell means increasingthe viability of a cell. In some embodiments, increasing the viabilityof a cell means: increasing the length of time the cell survives (e.g.as compared to an identical or similar unmodulated cell of the sametype), increasing the rate or amount of cellular proliferation (e.g. ascompared to an identical or similar unmodulated cell of the same type),or a combination thereof

In some embodiments, increasing the viability of a cell comprisescontacting a cell with an agent that increases the nucleic concentrationof a Myc polypeptide. In some embodiments, increasing the viability of acell comprises contacting a cell with: an exogenous Myc polypeptide, anagent that increases the expression of a MYC gene, and an agent thatincreases the activity of a Myc polypeptide, or a combination thereof(the “MYC Increasing Agent”). In some embodiments, the MYC IncreasingAgent is a small molecule, a peptide, an antibody, or a combinationthereof

In some embodiments, a method of increasing the viability of a cellcomprises contacting a cell with the MYC Increasing Agent. In someembodiments, the MYC Increasing Agent is an exogenous Myc polypeptide.In some embodiments, the MYC Increasing Agent is a fusion peptidecomprising (a) a transporter peptide sequence; (b) a MYC sequence; andoptionally (c) one or more molecules that link the transporter peptidesequence and the MYC sequence. In some embodiments, the MYC IncreasingAgent is a fusion peptide comprising (a) a TAT sequence; and (b) a MYCsequence.

In some embodiments, a method of increasing the viability of a cellcomprises contacting a cell with the MYC Increasing Agent. In someembodiments, the MYC Increasing Agent is an antagonist of a MAD-1 gene.In certain instances, the down-regulation of a MAD-1 gene and/orpolypeptide upregulates the expression of a MYC gene and/or apolypeptide encoded by a MYC gene.

In some embodiments, a method of increasing the viability of a cellcomprises contacting a cell with the MYC Increasing Agent. In someembodiments, the MYC Increasing Agent is an antagonist of a Mxi-1 gene.In certain instances, the down-regulation of a Mxi-1 gene and/orpolypeptide upregulates the expression of a MYC gene and/or apolypeptide encoded by a MYC gene.

In some embodiments, a method of increasing the viability of a cellfurther comprises identifying the MYC Increasing Agent. In someembodiments, the method of identifying the MYC Increasing Agentcomprises identifying an agent that reverses anergy in a B-cell. In someembodiments, the method of identifying the MYC Modulating Agentcomprises identifying an agent that increases the viability of afactor-dependent cell in the absence of a required growth factor. Insome embodiments, the method of identifying the MYC Increasing Agentcomprises identifying an agent that induces expression of a reportergene under the control of a MYC responsive promoter (e.g. an E-boxsequence).

In some embodiments, following contact with the MYC Increasing Agent theviability of a cell is increased by about 1 to about 20 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, following contact with the MYC Increasing Agent theviability of a cell is increased by about 1 to about 15 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, following contact with the MYC Increasing Agent theviability of a cell is increased by about 1 to about 10 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, following contact with the MYC Increasing Agent theviability of a cell is increased by about 1 to about 5 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, following contact with the MYC Increasing Agent theviability of a cell is increased by about 1 to about 4 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, following contact with the MYC Increasing Agent theviability of a cell is increased by 1 to about 2 times (e.g. as comparedto an identical or similar unmodulated cell of the same type).

In some embodiments, the cell is a T-cell. In some embodiments, theT-cell is a memory T-cell. In some embodiments, modulating the viabilityof a T-cell means extending the life-span of a T-cell. In someembodiments, extending the life span of a memory T-cell results in ahigher concentration of memory T-cells in a body. In certain instances,the higher concentration of memory T-cells results in an acceleratedprimary immune response to antigen. In certain instances, theup-regulation of a MYC gene results in a decrease in anergic T-cells. Incertain instances, the decrease in anergic T-cells results in anaccelerated primary immune response to antigen. In certain instances,the up-regulation of a MYC gene results in a decrease in the time ittakes a T-cell to activate in response to an antigen. In certaininstances, the decrease in the time it takes a T-cell to activate inresponse to an antigen results in an accelerated primary immune responseto antigen.

Decreasing the Viability of a Cell

In some embodiments, modulating the viability of a cell means decreasingthe viability of a cell. In some embodiments, decreasing the viabilityof a cell means: decreasing the length of time the cell survives anddecreasing the rate or amount of cellular proliferation (e.g., ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, decreasing the viability of a cell means decreasingthe length of time the cell survives and decreasing the rate or amountof cellular proliferation (e.g. as compared to an identical or similarunmodulated cell of the same type).

In some embodiments, decreasing the viability of a cell comprisescontacting a cell with an agent that decreases the nucleic concentrationof MYC. In some embodiments, decreasing the viability of a cellcomprises contacting a cell with: an agent that decreases the expressionof a MYC gene, an agent that decreases the activity of a Mycpolypeptide, or a combination thereof (the “MYC Decreasing Agent”). Insome embodiments, the MYC Decreasing Agent is a small molecule, abiologic, a peptide, an antibody, or a combination thereof.

In some embodiments, a method of decreasing the viability of a cellcomprises contacting a cell with the MYC Decreasing Agent. In someembodiments, the MYC Decreasing Agent is an agonist of a MAD-1 gene, aMAD-1 polypeptide, or a combination thereof. In certain instances, theup-regulation of a MAD-1 gene and/or polypeptide down-regulates theexpression of a MYC gene and/or a polypeptide encoded by a MYC gene. Insome embodiments, a method of modulating the viability of a cellcomprises contacting a cell with the MYC Decreasing Agent.

In some embodiments, a method of decreasing the viability of a cellcomprises contacting a cell with the MYC Decreasing Agent. In someembodiments, the MYC Decreasing Agent is: Mxi-1an agonist of a Mxi-1gene and a Mxi-1 polypeptide, or a combination thereof. In certaininstances, the up-regulation of a Mxi-1 gene and/or polypeptidedown-regulates the expression of a MYC gene and/or a polypeptide encodedby a MYC gene.

In some embodiments, the method of identifying the MYC Decreasing Agentcomprises identifying an agent that decreases the viability of afactor-dependent cell when the factor-dependent cell is contacted withthe necessary growth factor. In some embodiments, the method ofidentifying the MYC Decreasing Agent comprises identifying an agent thatinhibits expression of a reporter gene under the control of a MYCresponsive promoter (e.g. an E-box sequence).

In some embodiments, the viability of a cell is decreased by about 1 toabout 25 times (e.g. as compared to an identical or similar unmodulatedcell of the same type). In some embodiments, the viability of a cell isdecreased by about 1 to about 20 times (e.g. as compared to an identicalor similar unmodulated cell of the same type). In some embodiments, theviability of a cell is decreased by about 1 to about 15 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, the viability of a cell is decreased by about 1 toabout 10 times (e.g. as compared to an identical or similar unmodulatedcell of the same type). In some embodiments, the viability of a cell isdecreased by about 1 to about 5 times (e.g. as compared to an identicalor similar unmodulated cell of the same type). In some embodiments, theviability of a cell is decreased by about 1 to about 4 times (e.g. ascompared to an identical or similar unmodulated cell of the same type).In some embodiments, the viability of a cell is decreased by about 1 toabout 2 times (e.g. as compared to an identical or similar unmodulatedcell of the same type).

In some embodiments, the cell is a T-cell. In some embodiments, theT-cell is a memory T-cell. In certain instances, the down -regulation ofa MYC gene results in a decreased life-span for the T-cell. In certaininstances, the decreased life span of a memory T-cell results in a lowerconcentration of memory T-cells in a body. In certain instances, thelower concentration of memory T-cells results in a decelerated primaryimmune response to antigen, treatment of an autoimmune disorder, and/orimmunosuppression. In certain instances, the down-regulation of a MYCgene results in an increase in anergic T-cells. In certain instances,the increase in anergic T-cells results in a decelerated primary immuneresponse to antigen, treatment of an autoimmune disorder, and/orimmunosuppression. In certain instances, the down-regulation of a MYCgene results in an increase in the time it takes a T-cell to activate inresponse to an antigen. In certain instances, the increase in the timeit takes a T-cell to activate in response to an antigen results in adecelerated primary immune response to antigen, treatment of anautoimmune disorder, and/or immunosuppression.

Methods of Modulating an Immune Response

Disclosed herein, in certain embodiments, is a method of modulating animmune response. In some embodiments, the immune response is an adaptiveimmune response (e.g. memory B-cells and memory T-cells). In someembodiments, modulating an immune response means increasing (e.g.,inducing, supplementing, amplifying) an immune response. In someembodiments, modulating an immune response means decreasing (e.g.,inhibiting or suppressing) an immune response. In some embodiments, themodulation occurs in vitro. In some embodiments, the modulation occursin vivo.

In some embodiments, a method of modulating an immune response comprisesmodulating the viability of a lymphocyte. In some embodiments, thelymphocyte is a T-cell. In some embodiments, the T-cell is a CD4+T-cell. In some embodiments, the T-cell is a memory T-cell.

In some embodiments, modulating an immune response comprises modulatingthe expression of a MYC gene, and/or the activity of a Myc polypeptide.In some embodiments, modulating the expression of a MYC gene, and/or theactivity of a Myc polypeptide directly or indirectly results in themodulation of a gene or polypeptide regulated by MYC. In someembodiments, the gene or polypeptide directly or indirectly regulated bya MYC transcription factor is neurogranin, MEF-2c, gfi-1, cyclin D2,CDK4, Egr-2, Nab-2, TGIF, NF-kB p105, Carma-1, Al or Bc1-2.

In some embodiments, the subject is a human.

Inducing an Immune Response

In some embodiments, modulating an immune response means increasing(e.g., inducing, supplementing, amplifying) an immune response. In someembodiments, increasing an immune response includes increasing thelifespan of a T-cell, increasing the rate of proliferation by a T-cell,increasing the rate at which a memory T-cell responds to an antigen,decreasing the number of anergic cells, and/or increasing the rate atwhich a cell ends anergy (e.g. as compared to an identical or similarunmodulated cell of the same type).

In some embodiments, increasing an immune response comprisesadministering to an individual in need thereof an agent that increasesthe nucleic concentration of MYC. In some embodiments, increasing animmune response comprises administering to an individual in needthereof: exogenous MYC, an agent that increases the expression of a MYCgene, and an agent that increases the activity of MYC (the “MYCIncreasing Agent”). In some embodiments, the MYC Increasing Agent is asmall molecule, a biologic, a peptide, an antibody, or a combinationthereof.

In some embodiments, a method of increasing an immune response comprisescontacting a cell with the MYC Increasing Agent. In some embodiments,the MYC Increasing Agent is an exogenous Myc polypeptide. In someembodiments, the MYC Increasing Agent is a fusion peptide comprising (a)a transporter peptide sequence; (b) a MYC sequence; and optionally (c)one or more molecules that link the transporter peptide sequence and theMYC sequence. In some embodiments, the MYC Increasing Agent is a fusionpeptide comprising (a) a TAT sequence; and (b) a MYC sequence.

In some embodiments, a method of increasing an immune response comprisescontacting a cell with the MYC Increasing Agent. In some embodiments,the MYC Increasing Agent is an antagonist of a MAD-1 gene. In certaininstances, the down-regulation of a MAD-1 gene and/or polypeptideupregulates the expression of a MYC gene and/or a polypeptide encoded bya MYC gene.

In some embodiments, a method of increasing an immune response comprisescontacting a cell with the MYC Increasing Agent. In some embodiments,the MYC Increasing Agent is an antagonist of a Mxi-1 gene. In certaininstances, the down-regulation of a Mxi-1 gene and/or polypeptideupregulates the expression of a MYC gene and/or a polypeptide encoded bya MYC gene.

In some embodiments, following contact with the MYC Increasing Agent, animmune response is increased by more than 1 to about 20 times (e.g. ascompared to an identical or similar unmodulated immune response of thesame type). In some embodiments, following contact with the MYCIncreasing Agent an immune response is increased by more than 1 to about15 times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type). In some embodiments, following contact withthe MYC Increasing Agent an immune response is increased by more than 1to about 10 times (e.g. as compared to an identical or similarunmodulated immune response of the same type). In some embodiments,following contact with the MYC Increasing Agent an immune response isincreased by more than 1 to about 5 times (e.g. as compared to anidentical or similar unmodulated immune response of the same type). Insome embodiments, following contact with the MYC Increasing Agent animmune response is increased by more than 1 to about 4 times (e.g. ascompared to an identical or similar unmodulated immune response of thesame type). In some embodiments, following contact with the MYCIncreasing Agent an immune response is increased by more than 1 to about2 times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type).

In some embodiments, a method of increasing an immune response comprisesadministering a MYC Increasing Agent to an individual in need thereof.In some embodiments, following administration of the MYC IncreasingAgent to an individual in need thereof, an immune response is increasedby more than 1 to about 10 times (e.g. as compared to an identical orsimilar unmodulated immune response of the same type). In someembodiments, following administration of the MYC Increasing Agent to anindividual in need thereof, an immune response is increased by more than1 to about 20 times (e.g. as compared to an identical or similarunmodulated immune response of the same type). In some embodiments,following administration of the MYC Increasing Agent to an individual inneed thereof, an immune response is increased by more than 1 to about 15times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type). In some embodiments, followingadministration of the MYC Increasing Agent to an individual in needthereof, an immune response is increased by more than 1 to about 10times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type). In some embodiments, followingadministration of the MYC Increasing Agent to an individual in needthereof, an immune response is increased by more than 1 to about 5 times(e.g. as compared to an identical or similar unmodulated immune responseof the same type). In some embodiments, following administration of theMYC Increasing Agent to an individual in need thereof, an immuneresponse is increased by more than 1 to about 4 times (e.g. as comparedto an identical or similar unmodulated immune response of the sametype). In some embodiments, following administration of the MYCIncreasing Agent to an individual in need thereof, an immune response isincreased by more than 1 to about 2 times (e.g. as compared to anidentical or similar unmodulated immune response of the same type).

In some embodiments, a method of increasing an immune response furthercomprises co-administering a vaccine. In some embodiments, the vaccineis administered before, after, or simultaneously with a MYC IncreasingAgent. In some embodiments, the MYC Increasing Agent stimulates theimmune system and increases the response of the immune system to avaccine. In some embodiments, the MYC Increasing Agent augments animmune response. In some embodiments, the MYC Increasing Agent actssynergistically with the vaccine. In some embodiments, the agent is avaccine adjuvant.

In some embodiments, a vaccine comprises dead microorganisms, attenuatedmicroorganisms, toxoids, subunits of the pathogen, nucleic acids, orcombinations thereof. In some embodiments, the vaccine is a vaccine forhepatitis A; hepatitis B; polio; measles; mumps; rubella; diphtheria;pertussis; tetanus; influenza; varicella zoster virus; rotavirus;influenza; meningococcal disorder; pneumonia; smallpox; cholera; bubonicplague; yellow fever; tuberculosis: human paplomavirus (HPV); malaria;leishmania; Candida albicans; an allergen; or combinations thereof. Insome embodiments, the vaccine is a vaccine for a cancer (e.g. FollicularB-cell Non-Hodgkin's Lymphoma, prostate cancer, multiple myeloma, kidneycancer, cutaneous melanoma, and ocular melanoma). In some embodiments, acancer vaccine is a patient-specific vaccine (e.g. the vaccine comprisesa patient's own tumor cells). In some embodiments, a cancer vaccinecomprises Prostate Specific Antigen (PSA). In some embodiments, a cancervaccine comprises sialyl Tn (STn). In some embodiments, a cancer vaccinecomprises Heat Shock Proteins (HSPs) (e.g., gp96). In some embodiments,a cancer vaccine comprises ganglioside molecules (e.g., GM2, GD2, andGD3). In some embodiments, a cancer vaccine comprises carcinoembryonicantigen (CEA). In some embodiments, a cancer vaccine comprises MART-1(also known as Melan-A). In some embodiments, a cancer vaccine comprisestyrosinase. In some embodiments, the vaccine is a DNA vaccine.

In some embodiments, the vaccine comprises an antigenic moiety. In someembodiments, the antigenic moiety is a toxoid, a peptide, a nucleic acidsequence, a polysaccharide, or a combination thereof In someembodiments, the antigenic moiety is derived from a pathogen selectedfrom: hepatitis A; hepatitis B; polio; measles; mumps; rubella;diphtheria; pertussis; tetanus; influenza; varicella zoster virus;rotavirus; meningococcal; pneumonia; smallpox; cholera; bubonic plague;yellow fever; tuberculosis; human papillomavirus; or combinationsthereof In some embodiments, the antigenic moiety is derived aneoplastic cell. In some embodiments, the antigenic moiety is a nucleicacid or a polymer of nucleic acids.

In some embodiments, increasing an immune response in an individualreceiving vaccination against an antigen results in an increase in theviability (and thus concentration) of memory T-cells, B-cells, or acombination thereof against that antigen. In certain instances,increasing an immune response in an individual receiving vaccinationagainst an antigen results in accelerated activation of the T-cell bythe antigen. In certain instances, increasing an immune response in anindividual receiving vaccination against an antigen results in adecrease in anergic T-cells.

In some embodiments, a method of increasing an immune response furthercomprises identifying the MYC Increasing Agent. In some embodiments, themethod of identifying the MYC Increasing Agent comprises identifying anagent that reverses anergy in a B-cell. In some embodiments, the methodof identifying the MYC Increasing Agent comprises identifying an agentthat increases the viability of a factor-dependent cell in the absenceof a required growth factor. In some embodiments, the method ofidentifying the MYC Increasing Agent comprises identifying an agent thatinduces expression of a reporter gene under the control of a MYCresponsive promoter (e.g. an E-box sequence).

Decreasing an Immune Response

In some embodiments, modulating an immune response means decreasing(e.g., inhibiting or suppressing) an immune response. In someembodiments, decreasing an immune response includes decreasing thelifespan of a T-cell, decreasing proliferation by a T-cell, decreasingthe rate at which a memory T-cell responds to an antigen, increasing thenumber of anergic cells, and/or decreasing the rate at which a cell endsanergy (e.g. as compared to an identical or similar unmodulated cell ofthe same type).

In some embodiments, decreasing an immune response comprisesadministering to an individual in need thereof an agent that decreasesthe nucleic concentration of MYC. In some embodiments, decreasing animmune response comprises administering to an individual in needthereof: an agent that decreases the expression of a MYC gene and anagent that decreases the activity of a Myc polypeptide (the “MYCDecreasing Agent”). In some embodiments, the MYC Decreasing Agent is asmall molecule, a biologic, a peptide, an antibody, or a combinationthereof.

In some embodiments, a method of decreasing an immune response comprisescontacting a cell with the MYC Decreasing Agent. In some embodiments,the MYC Decreasing Agent is: an agonist of a MAD-1 gene, a MAD-1polypeptide, or a combination thereof. In certain instances, theup-regulation of a MAD-1 gene and/or polypeptide down-regulates theexpression of a MYC gene and/or a polypeptide encoded by a MYC gene.

In some embodiments, a method of decreasing an immune response comprisescontacting a cell with the MYC Decreasing Agent. In some embodiments,the MYC Decreasing Agent is: Mxi-1an agonist of a Mxi-1 gene and a Mxi-1polypeptide, or a combination thereof. In certain instances, theup-regulation of a Mxi-1 gene and/or polypeptide down-regulates theexpression of a MYC gene and/or a polypeptide encoded by a MYC gene.

In some embodiments, following contact with the MYC Decreasing Agent animmune response is decreased by more than 1 to about 25 times (e.g. ascompared to an identical or similar unmodulated immune response of thesame type). In some embodiments, following contact with the MYCDecreasing Agent an immune response is decreased by more than 1 to about20 times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type). In some embodiments, following contact withthe MYC Decreasing Agent an immune response is decreased by more than 1to about 15 times (e.g. as compared to an identical or similarunmodulated immune response of the same type). In some embodiments,following contact with the MYC Decreasing Agent an immune response isdecreased by more than 1 to about 10 times (e.g. as compared to anidentical or similar unmodulated immune response of the same type). Insome embodiments, following contact with the MYC Decreasing Agent animmune response is decreased by more than 1 to about 5 times (e.g. ascompared to an identical or similar unmodulated immune response of thesame type). In some embodiments, following contact with the MYCDecreasing Agent an immune response is decreased by more than 1 to about4 times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type). In some embodiments, following contact withthe MYC Decreasing Agent an immune response is decreased by more than 1to about 2 times (e.g. as compared to an identical or similarunmodulated immune response of the same type).

In some embodiments, a method of decreasing an immune response comprisesadministering a MYC Decreasing Agent to an individual in need thereof.In some embodiments, following administration of the MYC DecreasingAgent to an individual in need thereof an immune response is increasedby more than 1 to about 25 times (e.g. as compared to an identical orsimilar unmodulated immune response of the same type). In someembodiments, following administration of the MYC Decreasing Agent to anindividual in need thereof an immune response is increased by more than1 to about 20 times (e.g. as compared to an identical or similarunmodulated immune response of the same type). In some embodiments,following administration of the MYC Decreasing Agent to an individual inneed thereof an immune response is increased by more than 1 to about 15times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type). In some embodiments, followingadministration of the MYC Decreasing Agent to an individual in needthereof an immune response is increased by more than 1 to about 10 times(e.g. as compared to an identical or similar unmodulated immune responseof the same type). In some embodiments, following administration of theMYC Decreasing Agent to an individual in need thereof an immune responseis increased by more than 1 to about 5 times (e.g. as compared to anidentical or similar unmodulated immune response of the same type). Insome embodiments, following administration of the MYC Decreasing Agentto an individual in need thereof an immune response is increased by morethan 1 to about 4 times (e.g. as compared to an identical or similarunmodulated immune response of the same type). In some embodiments,following administration of the MYC Decreasing Agent to an individual inneed thereof an immune response is increased by more than 1 to about 2times (e.g. as compared to an identical or similar unmodulated immuneresponse of the same type).

In some embodiments, a method of decreasing an immune response comprisesadministering the MYC Decreasing Agent to an individual with anautoimmune disorder. In some embodiments, the MYC Decreasing Agentdecreases an immune response. In certain instances, decreasing an immuneresponse in an individual with an autoimmune disorder ameliorates and/orprevents an immune response against self antigens by the subject'simmune system. In some embodiments, the autoimmune disorder isCastleman's Disorder, lupus, multiple sclerosis, scleroderma pigmentosa,Autoimmune Lymphoproliferative Syndrome (ALPS), myesthenia gravis,diabetes, asthma, rheumatoid arthritis, vitiligo, diGeorge's syndrome,Grave's disorder, Crohn's disorder, inflammatory bowel disorder,colitis, orchitis, scleroderma pigmentosa, uveitis, Post-TransplantLymphoproliferative Disorder (PTLD), or Autoimmune Disorder-AssociatedLymphadenopathy (ADAL).

In some embodiments, a method of decreasing an immune response comprisesadministering the MYC Decreasing Agent to an individual that isreceiving, will receive, or has received an organ or bone marrowtransplant (the “Transplant”). In some embodiments, the MYC DecreasingAgent decreases an immune response. In certain instances, decreasing animmune response in an individual that is receiving, will receive, or hasreceived an organ transplant, or a bone marrow transplant amelioratesand/or prevents an immune response against the Transplant.

In some embodiments, the method of identifying the MYC Decreasing Agentcomprises identifying an agent that decreases the viability of afactor-dependent cell when the factor-dependent cell is contacted withthe necessary growth factor. In some embodiments, the method ofidentifying the MYC Decreasing Agent comprises identifying an agent thatinhibits expression of a reporter gene under the control of a MYCresponsive promoter (e.g. an E-box sequence).

Methods of Modulating a Disorder Characterized by the AberrantExpression of a MYC Gene or the Aberrant Activity of a MYC Polypeptide

Disclosed herein, in certain embodiments, are methods of treating adisorder characterized by an over-expression of MYC or an excess of Mycpolypeptide activity. Further disclosed herein, in certain embodiments,are methods of treating a disorder characterized by an under-expressionof MYC or a dearth of Myc polypeptide activity. In some embodiments, themethod comprises administering to an individual in need thereof aneffective amount of an agent that modulates the expression of a MYCgene, and/or the activity of a Myc polypeptide.

Disclosed herein, in certain embodiments, are methods of treating adisorder characterized by an under-expression of MYC or a dearth of Mycpolypeptide activity. In some embodiments, the method comprisesadministering to an individual in need thereof an effective amount of aMYC Increasing Agent. In some embodiments, the MYC Increasing Agent is asmall molecule, a biologic, a peptide, an antibody, or a combinationthereof.

In some embodiments, the method comprises administering to an individualin need thereof an effective amount of a MYC Increasing Agent. In someembodiments, the MYC Increasing Agent is an exogenous Myc polypeptide.In some embodiments, the MYC Increasing Agent is a fusion peptidecomprising (a) a transporter peptide sequence; (b) a MYC sequence; andoptionally (c) one or more molecules that link the transporter peptidesequence and the MYC sequence. In some embodiments, the MYC IncreasingAgent is a fusion peptide comprising (a) a TAT sequence; and (b) a MYCsequence.

In some embodiments, the method comprises administering to an individualin need thereof an effective amount of a MYC Increasing Agent. In someembodiments, the MYC Increasing Agent is an antagonist of a MAD-1 gene.In certain instances, the down-regulation of a MAD-1 gene and/orpolypeptide upregulates the expression of a MYC gene and/or apolypeptide encoded by a MYC gene.

In some embodiments, the method comprises administering to an individualin need thereof an effective amount of a MYC Increasing Agent. In someembodiments, the MYC Increasing Agent is an antagonist of a Mxi-1 gene.In certain instances, the down-regulation of a Mxi-1 gene and/orpolypeptide upregulates the expression of a MYC gene and/or apolypeptide encoded by a MYC gene.

Disclosed herein, in certain embodiments, are methods of treating adisorder characterized by an over-expression of MYC or an excess of Mycpolypeptide activity. In some embodiments, the method comprisesadministering to an individual in need thereof an effective amount of aMYC Decreasing Agent. In some embodiments, the MYC Decreasing Agent is asmall molecule, a biologic, a peptide, an antibody, or a combinationthereof

In some embodiments, the method comprises administering to an individualin need thereof an effective amount of a MYC Decreasing Agent. In someembodiments, the MYC Decreasing Agent is: an agonist of a MAD-1 gene, aMAD-1 polypeptide, or a combination thereof. In certain instances, theup-regulation of a MAD-1 gene and/or polypeptide down-regulates theexpression of a MYC gene and/or a polypeptide encoded by a MYC gene.

In some embodiments, the method comprises administering to an individualin need thereof an effective amount of a MYC Decreasing Agent. In someembodiments, a method of modulating the viability of a cell comprisescontacting a cell with the MYC Decreasing Agent. In some embodiments,the MYC Decreasing Agent is: Mxi-1an agonist of a Mxi-1 gene and a Mxi-1polypeptide, or a combination thereof. In certain instances, theup-regulation of a Mxi-1 gene and/or polypeptide down-regulates theexpression of a MYC gene and/or a polypeptide encoded by a MYC gene.

In some embodiments, an indication that an individual over-expresses MYCor that a Myc polypeptide is excessively active is the development of anautoimmune disorder. In some embodiments, the autoimmune disorder isCastleman's Disease, lupus, multiple sclerosis, scleroderma pigmentosa,Autoimmune Lymphoproliferative Syndrome (ALPS), myesthenia gravis,diabetes, asthma, rheumatoid arthritis, vitiligo, diGeorge's syndrome,Grave's disease, Crohn's disease, inflammatory bowel disease, colitis,orchitis, scleroderma pigmentosa, uveitis, Post-TransplantLymphoproliferative Disease (PTLD), Autoimmune Disease-AssociatedLymphadenopathy (ADAL), rejection of an organ transplant, rejection of atissue transplant, or combinations thereof.

In some embodiments, an indication that an individual under-expressesMYC, or that a Myc polypeptide is minimally active or inactive is anincrease in the number of anergic B-cells and/or T-cells. In certaininstances, the expression of IgM, IgMa, IgMb, B220, CD21/35, CD23, CD24(HSA), CD40, CD69, CD80 and/or CD86 (B7-2) is down-regulated in anergicB-cells when compared to non-anergic B-cells. In certain instances,reversing (e.g. partially or fully) anergy results in an increase in theexpression of IgM, IgMa, IgMb, B220, CD21/35, CD23, CD24 (HSA), CD40,CD69, CD80 and/or CD86 (B7-2).

In some embodiments, a method of treating a disorder characterized byaberrant expression of a MYC gene or aberrant activity of a Mycpolypeptide comprises identifying an agent that reverses anergy in aB-cell. In some embodiments, a method of treating a disordercharacterized by aberrant expression of a MYC gene or aberrant activityof a Myc polypeptide comprises identifying an agent that increases theviability of a factor-dependent cell in the absence of a required growthfactor. In some embodiments, a method of treating a disordercharacterized by aberrant expression of a MYC gene or aberrant activityof a Myc polypeptide comprises identifying an agent that decreases theviability of a factor-dependent cell when the factor-dependent cell iscontacted with the necessary growth factor. In some embodiments, amethod of treating a disorder characterized by aberrant expression of aMYC gene or aberrant activity of a Myc polypeptide comprises identifyingan agent that induces expression of a reporter gene under the control ofa MYC responsive promoter (e.g. an E-box sequence). In some embodiments,a method of treating a disorder characterized by aberrant expression ofa MYC gene or aberrant activity of a Myc polypeptide comprisesidentifying an agent that inhibits expression of a reporter gene underthe control of a MYC responsive promoter (e.g. an E-box sequence).

Exogenous MYC

Disclosed herein, in certain embodiments, is a fusion peptide comprising(a) a transporter peptide sequence; and (b) a MYC sequence. In someembodiments, the fusion peptide is a peptide of Formula (I):

-   -   transporter peptide sequence —MYC sequence.

In some embodiments, a fusion peptide disclosed herein comprises (a) atransporter peptide sequence; (b) a MYC sequence; and (c) one or moremolecules that link the transporter peptide sequence and the MYCsequence. In some embodiments, the fusion peptide is a peptide ofFormula (II):

-   -   transporter peptide sequence—X—MYC sequence,        wherein —X— is molecule that links the transporter peptide        sequence and the MYC sequence. In some embodiments, —X— is an        amino acid. In some embodiments, —X— is at least one amino acid.

As used herein, a “transporter peptide” means a peptide sequence thatpromotes peptide penetration into cells and tissues. In someembodiments, a transporter peptide is TAT. In some embodiments, atransporter peptide is TAT_([48-57]). In some embodiments, a transporterpeptide is TAT_([57-48]).]

As used herein, a “MYC sequence” is a MYC amino acid peptide sequence.In some embodiments, the Myc polypeptide is a complete Myc polypeptidesequence. In some embodiments, the Myc polypeptide is a partial Mycpolypeptide sequence. In some embodiments, the MYC is c-MYC. In someembodiments, the Myc polypeptide sequence comprises SEQ ID NO. 1:

MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLE.

In some embodiments, a fusion peptide disclosed herein comprises (a)TAT, and (b) c-MYC. In some embodiments, a fusion peptide disclosedherein comprises (a) TAT_([48-57]), and (b) c-MYC. In some embodiments,a fusion peptide disclosed herein comprises (a) TAT_([57-48]), and (b)c-MYC.

In some embodiments, a fusion peptide disclosed herein comprises (a)TAT, (b) a linker amino acid, and (c) c-MYC. In some embodiments, afusion peptide disclosed herein comprises (a) TAT_([48-57]), (b) alinker amino acid, and (c) c-MYC. In some embodiments, a fusion peptidedisclosed herein comprises (a) TAT_([57-48]), (b) a linker amino acid,and (c) c-MYC.

In some embodiments, a fusion peptide disclosed herein further comprisesat least one amino acid sequence that facilitates purification of thefusion protein. In some embodiments, a fusion peptide disclosed hereincomprises a protein tag. In some embodiments, a fusion peptide disclosedherein comprises a polyhistidine tag. In some embodiments, a fusionpeptide disclosed herein comprises an epitope tag. In some embodiments,a fusion peptide disclosed herein comprises a polyhistidine tag and anepitope tag. In some embodiments, a fusion peptide disclosed hereincomprises a 6-histidine tag and a V5 epitope tag.

In some embodiments, the histidine tag is a 6-histidine tag. In someembodiments, the histidine tag comprises the sequence HHHHHH. In someembodiments, a histidine tag is added to a fusion protein disclosedherein by any suitable method. In some embodiments, a TAT-Mycpolypeptide sequence is cloned into an expression vector encoding apolyHis-tag. In some embodiments, a polyhistidine tag is added by PCR(i.e., the PCR primers comprise a polyhistine sequence).

In some embodiments, a fusion peptide disclosed herein further comprisesat least one protein tag. In some embodiments, a fusion peptidedisclosed herein comprises an epitope tag. In some embodiments, a fusionpeptide disclosed herein further comprises a V5 epitope tag. In someembodiments, the V5 tag comprises the amino acids: GKPIPNPLLGLDST. Insome embodiments, the V5 tag comprises the amino acids: IPNPLLGLD. Insome embodiments, a V5 tag is added to a fusion protein disclosed hereinby any suitable method. In some embodiments, a TAT-Myc polypeptidesequence is cloned into an expression vector encoding a V5 tag. In someembodiments, a V5 tag is added by PCR (i.e., the PCR primers comprise aV5 sequence).

In some embodiments, the amino acids are in the D formation. In someembodiments, the amino acids are in the L formation. In someembodiments, a first plurality of amino acids are in the D formation anda second plurality are in the L formation.

In some embodiments, the MYC Increasing Agent comprises SEQ ID NO. 2:

MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH.

Construction of a MYC-TAT Peptide

In some embodiments, a MYC-TAT fusion peptide disclosed herein isconstructed by any suitable method. In some embodiments, a nucleotidesequence encoding a MYC-TAT fusion peptide is generated by PCR. In someembodiments, a forward primer for a human MYC sequence comprises an inframe N-terminal 9-amino-acid sequence of the TAT protein transductiondomain (i.e., RKKRRQRRR). In some embodiments, a reverse primer for ahuman MYC sequence is designed to remove the stop codon. In someembodiments, the PCR product is cloned into any suitable expressionvector (hereinafter, p-TAT-MYC). In some embodiments, the expressionvector comprises a polyhistidine tag and a V5 tag.

Assays for the Identification of Agents Reversal of Anergy

Disclosed herein, in certain embodiments, is a method of identifying anagent that reverses anergy in a B-cell. In certain instances, an agentthat reverses anergy in a B-cell increases an immune response and/orincreases the viability of a cell. In certain instances, an agent thatreverses anergy in a B-cell up-regulates MYC (e.g. a MYC gene and/or aMyc polypeptide).

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises (a) contacting a plurality of anergicB-cells with an agent; and (b) following contacting the plurality ofcells with the agent, detecting and/or measuring the level of expressionof one or more cell surface markers (e.g. cell surface markers thepresence of which is indicative a non-anergic cell) in the cell culture.In some embodiments, the cell surface marker is IgM, IgMa, IgMb, B220,CD21/35, CD23, CD24 (HSA), CD40, CD69, CD80 and/or CD86 (B7-2).

In certain instances, the expression of IgM, IgMa, IgMb, B220, CD21/35,CD23, CD24 (HSA), CD40, CD69, CD80 and/or CD86 (B7-2) is down-regulatedin anergic B-cells when compared to non-anergic B-cells. In certaininstances, reversing (e.g. partially or fully) anergy results in anincrease in the expression of IgM, IgMa, IgMb, B220, CD21/35, CD23, CD24(HSA), CD40, CD69, CD80 and/or CD86 (B7-2).

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises identifying an agent as an agonist of animmune response if expression of a cell surface marker is upregulated(e.g. as compared to an anergic B-cell not contacted with an agent). Insome embodiments, expression of a cell surface marker is upregulated onactivated B-cells (when compared to the expression pattern on anergicB-cells) by about 10% to about 100% (e.g. at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent fromabout 10% to about 100%). In some embodiments, expression of a cellsurface marker is upregulated on activated B-cells (when compared to theexpression pattern on anergic B-cells) by about 30% to about 100% (e.g.at least 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 30% to about 100%). In some embodiments, expressionof a cell surface marker is upregulated on activated B-cells (whencompared to the expression pattern on anergic B-cells) by about 50% toabout 100% (e.g. at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percentfrom about 50% to about 100%). In some embodiments, expression of a cellsurface marker is upregulated on activated B-cells (when compared to theexpression pattern on anergic B-cells)by about 70% to about 100% (e.g.at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%,96%, 97%, 98%, or any other percent from about 70% to about 100%). Insome embodiments, expression of a cell surface marker is upregulated onactivated B-cells (when compared to the expression pattern on anergicB-cells) by about 80% to about 100% (e.g. at least 80%, 85%, 86%, 87%,88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent fromabout 80% to about 100%).

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises identifying an agent as an antagonist of animmune response if expression of a cell surface marker is downregulated(e.g. as compared to a non-anergic B-cell not contacted with an agent).In some embodiments, expression of a cell surface marker isdownregulated (e.g. as compared to a non-anergic B-cell not contactedwith an agent) by about 10% to about 100% (e.g. at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percentfrom about 10% to about 100%). In some embodiments, expression of a cellsurface marker is downregulated (e.g. as compared to a non-anergicB-cell not contacted with an agent) by about 30% to about 100% (e.g. atleast 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percentfrom about 30% to about 100%). In some embodiments, expression of a cellsurface marker is downregulated (e.g. as compared to a non-anergicB-cell not contacted with an agent) by about 50% to about 100% (e.g. atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%,92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 50% toabout 100%). In some embodiments, expression of a cell surface marker isdownregulated (e.g. as compared to a non-anergic B-cell not contactedwith an agent) by about 70% to about 100% (e.g. at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 70% to about 100%). In some embodiments, expressionof a cell surface marker is downregulated (e.g. as compared to anon-anergic B-cell not contacted with an agent) by about 80% to about100% (e.g. at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%,96%, 97%, 98%, or any other percent from about 80% to about 100%).

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell further comprises comparing (1) the level ofexpression of the cell surface marker observed in a plurality of anergicB-cells following contacting the plurality of cells with the agent to(2) the level of cell surface marker observed in a control. In someembodiments, the control is a plurality of anergic B-cells not contactedwith an agent.

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises obtaining an anergic B-cell from anysuitable source (e.g. a human, a mouse, a rat, or any mammal). In someembodiments, a method of identifying an agent that reverses anergy in aB-cell comprises obtaining an anergic B-cell from transgenic mice withthe phenotype BCR^(HEL)/sHEL. In certain instances, BCR^(HEL) miceexpress a mature B-cell receptor (BCR) for the antigen hen egg lysozyme(HEL). In certain instances, the HEL BCR is the dominant BCR on theB-cells of the transgenic mice. In certain instances, sHEL miceubiquitously express a transgene for the soluble form of HEL. In certaininstances, the expression of the HEL transgene is operably linked to ametallothionein promoter. In certain instances, the exposure to solubleHEL induces anergy in B-cells. U.S. Patent Application Pub. No.2008/0070256 is hereby incorporated by reference for the methods ofconstructing the murine models discussed above. In some embodiments, theanergic B-cells are obtained from the spleen, thymus, bone marrow, lymphnodes, or combinations thereof of the transgenic mice.

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises detecting and/or measuring the level ofexpression of one or more cell surface markers (e.g. CD86). In someembodiments, detecting and/or measuring the level of expression of oneor more cell surface markers comprises (a) contacting a plurality ofanergic B-cells (e.g. a control, or a plurality of anergic B-cellsfollowing contact with an agent) with antibodies to a cell surfacemarker (e.g. antibodies to CD86); (b) washing the cell culture (e.g.rinsing) with buffer (e.g. FACS buffer) after contact with theantibodies; and (c) detecting and/or measuring the amount ofantibody/cell surface marker complex. In some embodiments, theantibodies are purchased from a commercial supplier. In someembodiments, the antibodies are generated in-house. For methods ofgenerating antibodies, see Kohler et al., Nature, 256:495 (1975); U.S.Pat. No. 4,816,567; or Goding, Monoclonal Antibodies: Principles andPractice (Academic Press, 1986); Ward et al., Nature 341: 544-546(1989); Huse et al., Science 246: 1275-1281 (1989); McCafferty et al.,Nature 348: 552-554 (1990); Clackson et al., Nature, 352:624-628 (1991)Marks et al., J. Mol. Biol., 222:581-597 (1991) all of which are herebyincorporated by reference. In some embodiments, the cell culture isincubated on ice during the contact with the antibodies. In someembodiments, the antibody is isotopically-labeled, radio-labeled,fluorophore-labeled, or biotinylated. In some embodiments, thefluorophore is fluorescein. In certain instances, the cell surfacemarker/antibody complex is detectable and/or measurable in any suitablemanner (e.g. HPLC, fluorescence microscopy, confocal microscopy,microarray scanners, Surface Plasmon Resonance, infrared spectroscopy,or autoradiography).

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises detecting and/or measuring the level ofexpression of IgM. In some embodiments, detecting and/or measuring thelevel of expression of IgM comprises (a) contacting a cell culture (e.g.a control, or a plurality of anergic B-cells following contact with anagent) with an antigen (e.g. HEL) for a period of time sufficient toallow the antigen to bind to an IgM; (b) washing the cell culture (e.g.rinsing) with buffer (e.g. FACS buffer) after contacting the culturewith the antigen; and (c) detecting and/or measuring the amount ofantigen and/or antigen/IgM complex. In some embodiments, the antigen isbiotinylated. In some embodiments, the cell culture is further incubatedwith a marker having an affinity for biotin (the “Biotin Marker”). Insome embodiments, the Biotin Marker is avidin or streptavidin. In someembodiments, the Biotin Marker is isotopically-labeled, radio-labeled,fluorophore-labeled. In some embodiments, the fluorophore isphycoerythrin. In certain instances, the IgM/antigen/Biotin Markercomplex is detectable and/or measurable in any suitable manner (e.g.HPLC, fluorescence microscopy, confocal microscopy, microarray scanners,Surface Plasmon Resonance, infrared spectroscopy, or autoradiography).

In some embodiments, a method of identifying an agent that reversesanergy in a B-cell comprises detecting and/or measuring the level ofexpression of IgM. In some embodiments, detecting and/or measuring thelevel of expression of IgM comprises (a) contacting a cell culture (e.g.a control, or a plurality of anergic B-cells following contact with anagent) with an antigen (e.g. HEL) for a period of time sufficient toallow the antigen to bind to the IgM; (b) washing the cell culture (e.g.rinsing) with buffer (e.g. FACS buffer) after contacting the culturewith the antigen; and (c) detecting and/or measuring the amount ofantigen and/or antigen/IgM complex. In some embodiments, the culture iscontacted with dilutions of antibodies to the antigen (e.g. for a periodof time sufficient to allow the antibodies to bind to the antigen). Insome embodiments, the cell culture is incubated on ice during contactwith the antibodies. In some embodiments, the cell culture is washed(e.g. rinsed) with buffer (e.g. FACS buffer) after contact with theantibodies. In some embodiments, the antibody is isotopically-labeled,radio-labeled, fluorophore-labeled, or biotinylated. In someembodiments, the fluorophore is fluorescein. In certain instances, theantigen/antibody and/or IgM/antigen/antibody complex is detectableand/or measurable in any suitable manner (e.g. HPLC, fluorescencemicroscopy, confocal microscopy, microarray scanners, Surface PlasmonResonance, infrared spectroscopy, or autoradiography).

In some embodiments, the method further comprises detecting the presenceof and/or measuring the amount of the labeled antibody, or of a complexformed between the antibody and the HEL antigen. In certain instances,the HEL antigen/antibody complex is detectable and measurable in anysuitable manner (e.g. HPLC, fluorescence microscopy, confocalmicroscopy, microarray scanners, Surface Plasmon Resonance, infraredspectroscopy, or autoradiography).

Modulation of Cytokine Function

Disclosed herein, in certain embodiments, is a method of identifying anagent that induces (e.g., activates, increases, or supplements) animmune response. In some embodiments, the method comprises identifyingan agent that replaces and/or augments cytokine function (the “CytokineSupplementing Agent”). In certain instances, an agent that replacesand/or augments cytokine function in a factor-dependent cell increasesan immune response and/or increases the viability of a cell. In certaininstances, an agent that replaces and/or augments cytokine function in afactor-dependent cell up-regulates MYC (e.g. a MYC gene and/or a Mycpolypeptide).

In some embodiments, the method comprises (a) contacting a plurality offactor-dependent cells with an agent; and (b) following contacting thecell with the agent, detecting and/or measuring the level of expansionof the cell culture.

In some embodiments, the method further comprises identifying an agentas a Cytokine Supplementing Agent if, following contact with the agent,the cell culture expands (e.g. as compared to a cell culture notcontacted with an agent). In some embodiments, following contact withthe agent the cell culture expands (when compared to a cell culture notcontacted with an agent) by about 10% to about 100% (e.g. at least 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 10% to about 100%). In some embodiments, followingcontact with the agent the cell culture expands (when compared to a cellculture not contacted with an agent) by about 30% to about 100% (e.g. atleast 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percentfrom about 30% to about 100%). In some embodiments, following contactwith the agent the cell culture expands (when compared to a cell culturenot contacted with an agent) by about 50% to about 100% (e.g. at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%,94%, 95%, 96%, 97%, 98%, or any other percent from about 50% to about100%). In some embodiments, following contact with the agent the cellculture expands (when compared to a cell culture not contacted with anagent) by about 70% to about 100% (e.g. at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 70% to about 100%). In some embodiments, followingcontact with the agent the cell culture expands (when compared to a cellculture not contacted with an agent) by about 80% to about 100% (e.g. atleast 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%,or any other percent from about 80% to about 100%).

In some embodiments, a method of identifying an agent that that replacesand/or augments cytokine function further comprises comparing (1) thelevel of expansion observed in a plurality of factor-dependent cellsfollowing contacting the plurality of cells with an agent to (2) thelevel of expansion of a control. In some embodiments, the control is aplurality of factor-dependent cells not contacted with an agent.

In some embodiments, contacting a factor-dependent cell with an agentthat replaces and/or augments cytokine function results in theexpression of MYC being upregulated. In certain instances, theup-regulation of MYC results in factor-dependent cells that do notrequire a cytokine for viability. In certain instances, theup-regulation of MYC results in factor-dependent cells that proliferatein the absence of a cytokine. In certain instances, the up-regulation ofMYC results in factor-dependent cells that do not undergo apoptosis inthe absence of a cytokine.

In some embodiments, the factor-dependent cells are lymphoid cells (i.e.derived from cells of the lymphatic system). In some embodiments, thefactor-dependent cells is, by way of non-limiting example, IL-2-/-,IL-3-/-, IL-4 -/-, IL-5-/-, IL-6-/-, IL-7-/-, IL-8-/-, IL-9-/-,IL-10-/-, IL-11-/-, IL-12-/-, or any combinations thereof. In someembodiments, the factor-dependent cells are derived from a cell lineselected from, by way of non-limiting example: 2D6; 2E8; 10B10; ATH8;B13; BAF/3; BALM-4;BCL1; CT.4S; CT6; CTL44; CTLL-2; D1; D10; D36;Da;DS-1; Ea3.17; EL4; FL5.12; HT-2; IC-2; Kitt225; KT-3; L4; L138.8A;LBRM-33; LyD9; MC/9; MLA-144; Mono Mac 6; Nb2; NKC3; PB-1; Pno; PT-18;Ramos; Sez627; T10; T88; T1165; TALL-103; TF-1; TMD2; TS1; UT-7; XG-1;Y16; or a combination thereof. In some embodiments, the cells arederived from CTLL-2; or BAF/3 cell lines.

Disclosed herein, in certain embodiments, are methods of identifying anagent that inhibits and/or interferes with cytokine function (the“Cytokine Interfering Agent”). In certain instances, a CytokineInterfering Agent decreases an immune response and/or decreases theviability of a cell. In some embodiments, the method comprises (a)contacting a plurality of factor-dependent cells with a required growthfactor such that the cell culture expands; (b) contacting the pluralityof cells plurality of factor-dependent cells with an agent; and (c)following contacting the plurality of cells plurality offactor-dependent cells with the agent, detecting and/or measuring thelevel of expansion of the cell culture.

In some embodiments, the method further comprises identifying an agentas cytokine interfering agent if, following contact with the agent, thecell culture contracts (e.g. as compared to a cell culture not contactedwith an agent). In some embodiments, following contact with the agentthe cell culture contracts (when compared to a cell culture notcontacted with an agent) by about 10% to about 100% (e.g. at least 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 10% to about 100%). In some embodiments, followingcontact with the agent the cell culture contracts (when compared to acell culture not contacted with an agent) by about 30% to about 100%(e.g. at least 30%, 35%, 40%, 45%. 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any otherpercent from about 30% to about 100%). In some embodiments, followingcontact with the agent the cell culture contracts (when compared to acell culture not contacted with an agent) by about 50% to about 100%(e.g. at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about50% to about 100%). In some embodiments, following contact with theagent the cell culture contracts (when compared to a cell culture notcontacted with an agent) by about 70% to about 100% (e.g. at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, orany other percent from about 70% to about 100%). In some embodiments,following contact with the agent the cell culture contracts (whencompared to a cell culture not contacted with an agent) by about 80% toabout 100% (e.g. at least 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%,95%, 96%, 97%, 98%, or any other percent from about 80% to about 100%).

In some embodiments, a method of identifying an agent that that replacesand/or augments cytokine function further comprises comparing (1) thelevel of expansion observed in a plurality of factor-dependent cellsfollowing contacting the plurality of cells with an agent to (2) thelevel of expansion of a control. In some embodiments, the control is aplurality of factor-dependent cells not contacted with an agent.

In some embodiments, contacting a factor-dependent cell with an agentthat inhibits and/or interferes with cytokine function results in theexpression of MYC being down-regulated. In certain instances, the down-regulation of MYC results in factor-dependent cells that require acytokine for viability. In certain instances, the down -regulation ofMYC results in factor-dependent cells that do not proliferate in theabsence of a cytokine. In certain instances, the down -regulation of MYCresults in factor-dependent cells that undergo apoptosis in the absenceof a cytokine.

In some embodiments, the factor-dependent cells are lymphoid cells (i.e.derived from cells of the lymphatic system). In some embodiments, thefactor-dependent cells is, by way of non-limiting example, IL-2-/-,IL-3-/-, IL-4 -/-, IL-5-/-, IL-6-/-, IL-7-/-, IL-8-/-, IL-9-/-,IL-10-/-, IL-11-/-, IL-12-/-, or any combinations thereof. In someembodiments, the factor-dependent cells are derived from a cell lineselected from, by way of non-limiting example: 2D6; 2E8; 10B10; ATH8;B13; BAF/3; BALM-4;BCL1; CT.4S; CT6; CTL44; CTLL-2; D1; D10; D36;Da;DS-1; Ea3.17; EL4; FL5.12; HT-2; IC-2; Kitt225; KT-3; L4; L138.8A;LBRM-33; LyD9; MC/9; MLA-144; Mono Mac 6; Nb2; NKC3; PB-1; Pno; PT-18;Ramos; Sez627; T10; T88; T1165; TALL-103; TF-1; TMD2; TS1; UT-7; XG-1;Y16; or a combination thereof. In some embodiments, the cells arederived from CTLL-2; or BAF/3 cell lines.

Reporter Assay

Disclosed herein, in certain embodiments, is a method of identifying anagent that induces (e.g., activates, increases, or supplements) animmune response. In some embodiments, the method comprises identifyingan agent that modulates the function of a MYC encoded transcriptionfactor. In some embodiments, the method comprises (a) transforming aplurality of cells in a cell culture with a reporter construct, whereinthe reporter construct is a plasmid that comprises a reporter gene, andwherein the expression of the reporter gene is operably linked to one ormore E-box sequences; (b) contacting the plurality of cells plurality ofcells transformed with the reporter construct with an agent; and (c)following contacting the culture with an agent, detecting and/ormeasuring the level of expression of the reporter gene. In someembodiments, the reporter construct is obtained in any suitable manner(e.g. purchased from a vendor or fabricated in-house). For methodssuitable for fabricating a reporter construct and transforming cellswith a reporter construct see Molecular Cloning: A Laboratory Manual,second edition (Sambrook et al., 1989) and Molecular Cloning: ALaboratory Manual, third edition (Sambrook and Russel, 2001), jointlyreferred to herein as “Sambrook”); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987, including supplements through2001); Current Protocols in Nucleic Acid Chemistry John Wiley & Sons,Inc., New York, 2000) which are hereby incorporated by reference forsuch disclosure.

In some embodiments, a method of identifying an agent that modulates thefunction of a MYC encoded transcription factor further comprisescomparing (1) the level of expression of the reporter gene in aplurality of cells transformed with the reporter construct followingcontact with an agent to (2) the level of the level of expression of thereporter gene in a control.

In some embodiments, the cells are eukaryotic cells. In someembodiments, the eukaryotic cells are human cells. In some embodiments,the cells are human lymphoid cells. In some embodiments, the lymphoidcells are B-cells.

In some embodiments, the reporter construct comprises one or more E-boxsequences upstream of a reporter gene. In some embodiments, the one ormore E-box sequences are encoded in a myc-responsive promoter sequenceand/or are upstream of the myc-responsive promoter. As used here,“promoter sequence” means a nucleotide sequence upstream of a gene thatallows an RNA polymerase to transcribe the gene. As used herein,“myc-responsive promoter” means a promoter sequence to which atranscription factor encoded by MYC can bind. In some embodiments, themyc-responsive promoter is the ornithine decarboxylase promoter.

In some embodiments, the reporter gene is a β-galactosidase gene, aβ-lactamase gene, a horseradish peroxidase gene, an alkaline phosphatasegene, a thymidine kinase gene, a xanthine phosphoribotransferase gene, atyrosinase gene, a cytosine deaminase gene, an antibiotic resistancegene, or a gene having a fluorescent expression product. In someembodiments, the gene having a fluorescent expression product is aluciferase gene, or a green fluorescent polypeptide (GFP) gene.

In some embodiments, the method of identifying an agent that modulatesthe function of a MYC encoded transcription factor further comprisesdetecting the expression of and/or measuring the amount of expression ofthe reporter gene. In certain instances, the expression of the reportergene is detectable and measurable in any suitable manner (e.g.fluorescence microscopy).

In some embodiments, the cell culture further comprises serum. In someembodiments, the serum is fetal bovine serum (FBS); bovine serum; horseserum; human serum; chicken serum; goat serum; porcine serum; rabbitserum; sheep serum; a serum replacement (e.g. Serum Replacement 1(Sigma-Aldrich)); or a combination thereof. In some embodiments, thecell culture further comprises media. In some embodiments, the media is,by way of non-limiting example, phosphate buffered saline; Earle'sBalanced Salts; Hanks' Balanced Salts; Tyrode's Salts; derivativesthereof; or combinations thereof. In some embodiments, the growth factoris, by way of non-limiting example, a cytokine, epidermal growth factor,or platelet-derived growth factor. In some embodiments, the cytokine is,by way of non-limiting example, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, or IL-13. In certain instances, thechoice of growth factor is determined based on the type of cell used inthe method.

In some embodiments, the method further comprises contacting theplurality of cells with a mitogenic stimulant. In some embodiments, themitogenic stimulant is, by way of non-limiting example, an antigen, or amitogenic antibody. In some embodiments, the antigen is, by way ofnon-limiting example, phytohaemagglutinin (PHA), concanavalin A (conA),lipopolysaccharide (LPS), pokeweed mitogen (PWM), or combinationsthereof. In some embodiments, the mitogenic antibody is, by way ofnon-limiting example, an anti-IgM, an anti-CD40, or combinationsthereof. In some embodiments, the anti-IgM is anti-IgM-F(ab′)2. In someembodiments, the anti-CD40 is anti-CD40 IgM.

In vitro validation of agents

In some embodiments, any of the methods described herein furthercomprise validating in vitro an agent (e.g. an agent identifiedaccording to any method described herein) as a regulator of an immuneresponse. In some embodiments, a method of validating in vitro an agentas a regulator of an immune response comprises (a) contacting the agentwith a primary T-cell activation culture; (b) staining the culture foractivation markers; and (c) staining the culture 24 hours after contactwith the agent. In certain instances, the cell viability is detectableand measurable in any suitable manner (e.g. fluorescence microscopy,flow cytometry, FACS). In certain instances, an agent that up-regulatesan immune response will cause an increase in activation markers ascompared to a control (or the culture before contact with the agent). Incertain instances, an agent that down-regulates an immune response willcause a decrease in activation markers as compared to a control (or theculture before contact with the agent).

In some embodiments, any of the methods described herein furthercomprise validating in vitro an agent as a regulator of an immuneresponse. In some embodiments, a method of validating in vitro an agentas a regulator of an immune response comprises (a) contacting the agentwith a primary T-cell activation culture; (b) staining the culture withCFSE (carboxyfluorescein succinimidyl ester); and (c) staining theculture 72 hours after contact with the agent. In certain instances,CFSE indicates cell proliferation. In certain instances, a cellincorporates the CFSE. In certain instances, a portion (i.e. half) ofthe CFSE in a cell transfers to a daughter cell following division. Incertain instances, the amount of CFSE in cell decreases with eachdivision. In certain instances, the cell viability is detectable andmeasurable in any suitable manner (e.g. fluorescence microscopy, flowcytometry, FACS). In certain instances, an agent that up-regulates animmune response will cause an increase in cell proliferation as comparedto a control (or the culture before contact with the agent). In certaininstances, an agent that down-regulates an immune response will cause adecrease in cell proliferation as compared to a control (or the culturebefore contact with the agent).

In some embodiments, any of the methods described herein furthercomprise validating in vitro an agent as a regulator of an immuneresponse. In some embodiments, a method of validating in vitro an agentas a regulator of an immune response comprises (a) contacting the agentwith a primary T-cell activation culture; (b) staining the culture with7AAD (7-amino-actinomycin D); and (c) staining the culture 72 hoursafter contact with the agent. In certain instances, 7-AAD indicates cellviability. In certain instances, 7-AAD will stain cells with compromisedmembranes (i.e. unviable cells). In certain instances, 7-AAD will notstain cells with uncompromised membranes (i.e. viable cells). In certaininstances, the cell viability is detectable and measurable in anysuitable manner (e.g. fluorescence microscopy, flow cytometry, FACS). Incertain instances, an agent that up-regulates an immune response willcause an increase in cell viability as compared to a control (or theculture before contact with the agent). In certain instances, an agentthat down-regulates an immune response will cause a decrease in cellviability as compared to a control (or the culture before contact withthe agent). In vivo validation of agents

In some embodiments, any of the methods described herein furthercomprise validating in vivo an agent (e.g. an agent identified accordingto any method described herein) as a regulator of an immune response. Insome embodiments, a method of validating in vivo an agent as a regulatorof an immune response comprises (a) immunizing a mammal (e.g. a mouse,rat, rabbit) against an antigen (e.g. OVA peptides, NP-KHL) byadministering the antigen to a mouse; (b) administering the agent to themouse before, during, or after administration of the antigen; (c)obtaining a biological sample (e.g. blood, lymph, or serum) from themouse after administration of the antigen and agent; and (d) detectingor measuring an immune response (e.g. the rate of proliferation ofmemory B-cells and/or T-cells). In some embodiments, an immune responseis detected or measured by detecting and measuring antibody productionfollowing the immunization. In some embodiments, antibody production isassayed once-a-day every day for a week following immunization and thenonce-a-week thereafter. In some embodiments, an immune response isdetected or measured by detecting and measuring T-cell proliferationupon re-stimulation with the antigen. In some embodiments, T-cellproliferation is assayed one week after immunization. In certaininstances, an agent that up-regulates a primary immune response willcause an increase in antibody production and T-cell proliferation ascompared to a control (i.e. a mouse not administered the agent). Incertain instances, an agent that down-regulates a primary immuneresponse will cause a decrease in antibody production and T-cellproliferation as compared to a control (i.e. a mouse not administeredthe agent).

In some embodiments, any of the methods described herein furthercomprise validating in vivo an agent (e.g. an agent identified accordingto any method described herein) as a regulator of an immune response. Insome embodiments, a method of validating in vivo an agent as a regulatorof an immune response comprises (a) immunizing a mouse against anantigen (e.g. OVA peptides, NP-KHL) by administering the antigen to themouse; (b) administering the agent to the mouse before, during, or afteradministration of the antigen; (c) challenging the mouse with theantigen 3 to 6 months after inoculation; (d) obtaining a biologicalsample (e.g. blood, lymph, or serum) from the mouse following thechallenge; and (d) detecting or measuring an immune response. In someembodiments, an immune response is detected or measured by detecting andmeasuring antibody production following immunization. In someembodiments, antibody production is assayed once-a-day every day for aweek following challenge and then once-a-week thereafter. In someembodiments, an immune response is detected or measured by detecting andmeasuring T-cell proliferation upon re-stimulation with the antigen. Insome embodiments, T-cell proliferation is assayed one week afterchallenge. In certain instances, an agent that up-regulates a secondaryimmune response will cause an accelerated immune response (i.e. anincrease rate of antibody production and T-cell proliferation) followingchallenge with the antigen as compared to a control (i.e. a mouse notadministered the agent). In certain instances, an agent thatdown-regulates a secondary immune response will cause a deceleratedimmune response (i.e. a decreased rate of antibody production and T-cellproliferation) following challenge with the antigen as compared to acontrol (i.e. a mouse not administered the agent).

Agents

Disclosed herein, in certain embodiments, are agents that modulate animmune response (e.g. agents that reverse anergy, agents that modulatecytokine function, and/or agents that modulate the function of a MYCencoded transcription factor). In certain embodiments, the agent isuseful in administration to an individual in need thereof. In specificembodiments, the agent is any agent identified by any of the methodsdisclosed herein. In some embodiments, the agent is any agent thatmodulates an immune response. In some embodiments, the agent is a smallmolecule. In some embodiments, the agent is RNAi. In some embodiments,the agent is an agonist of MYC (e.g. a MYC gene and/or a Mycpolypeptide). In some embodiments, the agent is an antagonist of MYC(e.g. a MYC gene and/or a Myc polypeptide).

In some embodiments, the agent is identified as an agent that modulatesthe viability of a cell based on the agent's ability to reverse anergyin a B-cell. In some embodiments, the agent is identified as an agentthat that modulates the viability of a cell based on the agent's abilityto modulate the viability of factor-dependent cells. In someembodiments, the agent is identified as an agent that modulates theviability of a cell based on the agent's ability to induce expression ofa reporter gene.

In some embodiments, the modulation of the immune response comprises themodulation of the viability of a lymphocyte. In some embodiments, thelymphocyte is a T-cell. In some embodiments, the T-cell is a CD4+T-cell. In some embodiments, the T-cell is a memory T-cell.

In some embodiments, the viability of a lymphocyte following contactwith the agent is more than 1 to about 10 times greater than theviability of a lymphocyte that has not been contacted the agent. In someembodiments, the agent causes the up-regulation of a MYC gene in alymphocyte. In some embodiments, the modulation occurs in vivo. In someembodiments, the lymphocyte is a T-cell. In some embodiments, the T-cellis a memory T-cell. In certain instances, the up-regulation of a MYCgene results in an extended life-span for the T-cell. In certaininstances, the extended life span of a memory T-cell results in a higherconcentration of memory T-cells in a body. In certain instances, thehigher concentration of memory T-cells results in an accelerated primaryimmune response to antigen. In certain instances, the up-regulation of aMYC gene results in a decrease in anergic T-cells. In certain instances,the decrease in anergic T-cells results in an accelerated primary immuneresponse to antigen. In certain instances, the up-regulation of a MYCgene results in a decrease in the time it takes a T-cell to activate inresponse to an antigen. In certain instances, the decrease in the timeit takes a T-cell to activate in response to an antigen results in anaccelerated primary immune response to antigen.

In some embodiments, the agent is administered before, during, or afteradministration of a vaccine to an individual. In some embodiments, theagent stimulates the immune system and increases the response of theimmune system to a vaccine. In some embodiments, agent augments animmune response. In some embodiments, the agent acts synergisticallywith the vaccine. In some embodiments, the agent is a vaccine adjuvant.

In some embodiments, a vaccine comprises dead microorganisms, attenuatedmicroorganisms, toxoids, subunits of the pathogen, nucleic acids, orcombinations thereof. In some embodiments, the vaccine is a vaccine forhepatitis A; hepatitis B; polio; measles; mumps; rubella; diphtheria;pertussis; tetanus; influenza; varicella zoster virus; rotavirus;influenza; meningococcal disorder; pneumonia; smallpox; cholera; bubonicplague; yellow fever; tuberculosis: human paplomavirus; or combinationsthereof. In some embodiments, the vaccine is a vaccine for a cancer(e.g. follicular B-cell Non-Hodgkin's lymphoma, prostate cancer,multiple myeloma, kidney cancer, cutaneous melanoma, ocular melanoma,and other solid tumors, carcinomas and sarcomas). In some embodiments, acancer vaccine is a patient-specific vaccine (e.g. the vaccine comprisesa patient's own tumor cells). In some embodiments, a cancer vaccinecomprises Prostate Specific Antigen (PSA). In some embodiments, a cancervaccine comprises sialyl Tn (STn). In some embodiments, a cancer vaccinecomprises Heat Shock Proteins (HSPs) (e.g., gp96). In some embodiments,a cancer vaccine comprises ganglioside molecules (e.g., GM2, GD2, andGD3). In some embodiments, a cancer vaccine comprises carcinoembryonicantigen (CEA). In some embodiments, a cancer vaccine comprises MART-1(also known as Melan-A). In some embodiments, a cancer vaccine comprisestyrosinase. In some embodiments, the vaccine is a DNA vaccine.

In some embodiments, the comprises an antigenic moiety. In someembodiments, the antigenic moiety is a toxoid, a peptide, a nucleic acidsequence, a polysaccharide, or a combination thereof. In someembodiments, the antigenic moiety is derived from a pathogen selectedfrom: hepatitis A; hepatitis B; polio; measles; mumps; rubella;diphtheria; pertussis; tetanus; influenza; varicella zoster virus;rotavirus; meningococcal; pneumonia; smallpox; cholera; bubonic plague;yellow fever; tuberculosis; human papillomavirus; or combinationsthereof. In some embodiments, the antigenic moiety is derived aneoplastic cell. In some embodiments, the antigenic moiety is a nucleicacid or a polymer of nucleic acids.

In certain instances, up-regulating an immune response in an individualreceiving vaccination against an antigen results in an increase in theviability and thus concentration of memory T-cells against that antigen.In certain instances, up-regulating an immune response in an individualreceiving vaccination against an antigen results in acceleratedactivation of the T-cell by the antigen. In certain instances,up-regulating an immune response in an individual receiving vaccinationagainst an antigen results in a decrease in T-cells tolerant of theantigen.

In some embodiments, the viability of a lymphocyte administered theagent is more than 1 to about 25 times less than the viability of alymphocyte that has not been administered the agent. In someembodiments, the agent causes the down-regulation of a MYC gene in alymphocyte. In some embodiments, the modulation occurs in vivo. In someembodiments, the lymphocyte is a T-cell. In some embodiments, the T-cellis a memory T-cell. In certain instances, the down -regulation of a MYCgene results in a decreased life-span for the T-cell. In certaininstances, the decreased life span of a memory T-cell results in a lowerconcentration of memory T-cells in a body. In certain instances, thelower concentration of memory T-cells results in a decelerated primaryimmune response to antigen, treatment of an autoimmune disorder, and/orimmunosuppression. In certain instances, the down-regulation of a MYCgene results in an increase in anergic T-cells. In certain instances,the increase in anergic T-cells results in a decelerated primary immuneresponse to antigen, treatment of an autoimmune disorder, and/orimmunosuppression. In certain instances, the down-regulation of a MYCgene results in an increase in the time it takes a T-cell to activate inresponse to an antigen. In certain instances, the increase in the timeit takes a T-cell to activate in response to an antigen results in adecelerated primary immune response to antigen, treatment of anautoimmune disorder, and/or immunosuppression.

In some embodiments, the agent is administered to an individual with anautoimmune disorder. In some embodiments, the autoimmune disorder isCastleman's Disease, lupus, multiple sclerosis, scleroderma pigmentosa,Autoimmune Lymphoproliferative Syndrome (ALPS), myesthenia gravis,diabetes, asthma, rheumatoid arthritis, vitiligo, diGeorge's syndrome,Grave's disease, Crohn's disease, inflammatory bowel disease, colitis,orchitis, scleroderma pigmentosa, uveitis, Post-TransplantLymphoproliferative Disease (PTLD), or Autoimmune Disease-AssociatedLymphadenopathy (ADAL). In certain instances, down-regulating an immuneresponse in an individual with an autoimmune disorder ameliorates and/orprevents an immune response against self antigens by the subject'simmune system.

In some embodiments, the agent is administered to an individual that hasreceived an organ transplant, or a bone marrow transplant. In certaininstances, down-regulating an immune response in an individual that hasreceived an organ or bone marrow transplant ameliorates and/or preventsan immune response against the transplanted organ or bone marrow by thesubject's immune system.

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are compositions that modulatethe immune system in an individual in need thereof, wherein thecomposition comprises a therapeutically-effective amount of an agentidentified by any of the methods disclosed herein. In some embodiments,the agent is a small molecule. In some embodiments, the agent is RNAi.In some embodiments, the agent is a biological molecule (e.g., apeptide, a fusion peptide). In some embodiments, the agent is a fusionpeptide disclosed herein. In some embodiments, the agent is a fusionpeptide comprising (a) a transporter peptide sequence; and (b) a MYCsequence. In some embodiments, the agent is a fusion peptide of Formula(I):

-   -   transporter peptide sequence —MYC sequence.

In some embodiments, the agent is identified as an agent that modulatesthe viability of a cell based on the agent's ability to reverse anergyin a B-cell. In some embodiments, the agent is identified as an agentthat that modulates the viability of a cell based on the agent's abilityto modulate the viability of factor-dependent cells. In someembodiments, the agent is identified as an agent that modulates theviability of a cell based on the agent's ability to induce expression ofa reporter gene.

In some embodiments, the modulation of the immune response comprises themodulation of the viability of a lymphocyte. In some embodiments, thelymphocyte is a T-cell. In some embodiments, the T-cell is a CD4+T-cell. In some embodiments, the T-cell is a memory T-cell.

In some embodiments, the viability of a lymphocyte following contactwith the agent is more than 1 to about 10 times greater than theviability of a lymphocyte that has not been contacted the agent. In someembodiments, the agent causes the up-regulation of a MYC gene in alymphocyte. In some embodiments, the modulation occurs in vivo. In someembodiments, the lymphocyte is a T-cell. In some embodiments, the T-cellis a memory T-cell. In certain instances, the up-regulation of a MYCgene results in an extended life-span for the T-cell. In certaininstances, the extended life span of a memory T-cell results in a higherconcentration of memory T-cells in a body. In certain instances, thehigher concentration of memory T-cells results in an accelerated primaryimmune response to antigen. In certain instances, the up-regulation of aMYC gene results in a decrease in anergic T-cells. In certain instances,the decrease in anergic T-cells results in an accelerated primary immuneresponse to antigen. In certain instances, the up-regulation of a MYCgene results in a decrease in the time it takes a T-cell to activate inresponse to an antigen. In certain instances, the decrease in the timeit takes a T-cell to activate in response to an antigen results in anaccelerated primary immune response to antigen.

In some embodiments, the agent is administered before, during, or afteradministration of a vaccine to an individual. In some embodiments, theagent stimulates the immune system and increases the response of theimmune system to a vaccine. In some embodiments, the agent augments animmune response. In some embodiments, the agent acts synergisticallywith the vaccine. In some embodiments, the agent is a vaccine adjuvant.

In some embodiments, a vaccine comprises dead microorganisms, attenuatedmicroorganisms, toxoids, subunits of the pathogen, nucleic acids, orcombinations thereof. In some embodiments, the vaccine is a vaccine forhepatitis A; hepatitis B; polio; measles; mumps; rubella; diphtheria;pertussis; tetanus; influenza; varicella zoster virus; rotavirus;influenza; meningococcal disease; pneumonia; smallpox; cholera; bubonicplague; yellow fever; tuberculosis: human paplomavirus; or combinationsthereof. In some embodiments, the vaccine is a vaccine for a cancer(e.g. Follicular B-cell Non-Hodgkin's Lymphoma, prostate cancer,multiple myeloma, kidney cancer, cutaneous melanoma, and ocularmelanoma). In some embodiments, a cancer vaccine is a patient-specificvaccine (e.g. the vaccine comprises a patient's own tumor cells). Insome embodiments, a cancer vaccine comprises Prostate Specific Antigen(PSA). In some embodiments, a cancer vaccine comprises sialyl Tn (STn).In some embodiments, a cancer vaccine comprises Heat Shock Proteins(HSPs) (e.g., gp96). In some embodiments, a cancer vaccine comprisesganglioside molecules (e.g., GM2, GD2, and GD3). In some embodiments, acancer vaccine comprises carcinoembryonic antigen (CEA). In someembodiments, a cancer vaccine comprises MART-1 (also known as Melan-A).In some embodiments, a cancer vaccine comprises tyrosinase. In someembodiments, the vaccine is a DNA vaccine.

In some embodiments, the comprises an antigenic moiety. In someembodiments, the antigenic moiety is a toxoid, a peptide, a nucleic acidsequence, a polysaccharide, or a combination thereof. In someembodiments, the antigenic moiety is derived from a pathogen selectedfrom: hepatitis A; hepatitis B; polio; measles; mumps; rubella;diphtheria; pertussis; tetanus; influenza; varicella zoster virus;rotavirus; meningococcal; pneumonia; smallpox; cholera; bubonic plague;yellow fever; tuberculosis; human papillomavirus; or combinationsthereof. In some embodiments, the antigenic moiety is derived aneoplastic cell. In some embodiments, the antigenic moiety is a nucleicacid or a polymer of nucleic acids.

In certain instances, up-regulating an immune response in an individualreceiving vaccination against an antigen results in an increase in theviability and thus concentration of memory T-cells against that antigen.In certain instances, up-regulating an immune response in an individualreceiving vaccination against an antigen results in acceleratedactivation of the T-cell by the antigen. In certain instances,up-regulating an immune response in an individual receiving vaccinationagainst an antigen results in a decrease in T-cells tolerant of theantigen.

In some embodiments, the viability of a lymphocyte administered theagent is more than 1 to about 25 times less than the viability of alymphocyte that has not been administered the agent. In someembodiments, the agent causes the down-regulation of a MYC gene in alymphocyte. In some embodiments, the modulation occurs in vivo. In someembodiments, the lymphocyte is a T-cell. In some embodiments, the T-cellis a memory T-cell. In certain instances, the down -regulation of a MYCgene results in a decreased life-span for the T-cell. In certaininstances, the decreased life span of a memory T-cell results in a lowerconcentration of memory T-cells in a body. In certain instances, thelower concentration of memory T-cells results in a decelerated primaryimmune response to antigen, treatment of an autoimmune disorder, and/orimmunosuppression. In certain instances, the down-regulation of a MYCgene results in an increase in anergic T-cells. In certain instances,the increase in anergic T-cells results in a decelerated primary immuneresponse to antigen, treatment of an autoimmune disorder, and/orimmunosuppression. In certain instances, the down-regulation of a MYCgene results in an increase in the time it takes a T-cell to activate inresponse to an antigen. In certain instances, the increase in the timeit takes a T-cell to activate in response to an antigen results in adecelerated primary immune response to antigen, treatment of anautoimmune disorder, and/or immunosuppression.

In some embodiments, the agent is administered to an individual with anautoimmune disorder. In some embodiments, the autoimmune disorder isCastleman's Disease, lupus, multiple sclerosis, scleroderma pigmentosa,Autoimmune Lymphoproliferative Syndrome (ALPS), myesthenia gravis,diabetes, asthma, rheumatoid arthritis, vitiligo, diGeorge's syndrome,Grave's disease, Crohn's disease, inflammatory bowel disease, colitis,orchitis, scleroderma pigmentosa, uveitis, Post-TransplantLymphoproliferative Disease (PTLD), or Autoimmune Disease-AssociatedLymphadenopathy (ADAL). In certain instances, down-regulating an immuneresponse in an individual with an autoimmune disorder ameliorates and/orprevents an immune response against self antigens by the subject'simmune system.

In some embodiments, the agent is administered to an individual that hasreceived an organ transplant, or a bone marrow transplant. In certaininstances, down-regulating an immune response in an individual that hasreceived an organ or bone marrow transplant ameliorates and/or preventsan immune response against the transplanted organ or bone marrow by thesubject's immune system.

Formulations of Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions are formulated in aconventional manner using one or more physiologically acceptablecarriers including, e.g., excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which are suitablefor pharmaceutical use. In certain embodiments, proper formulation isdependent upon the route of administration chosen. A summary ofpharmaceutical compositions described herein is found, for example, inRemington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

A pharmaceutical composition, as used herein, refers to a mixture of acompound described herein, with other chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, and/or excipients. In certain instances, thepharmaceutical composition facilitates administration of the compound toan individual or cell. In certain embodiments of practicing the methodsof treatment or use provided herein, therapeutically effective amountsof compounds described herein are administered in a pharmaceuticalcomposition to an individual having a disorder, disease, or condition tobe treated. In specific embodiments, the individual is a human. Asdiscussed herein, the therapeutic compounds described herein are eitherutilized singly or in combination with one or more additionaltherapeutic agents.

In some embodiments, the pharmaceutical formulations described hereinare administered to an individual in any manner, including one or moreof multiple administration routes, such as, by way of non-limitingexample, oral, parenteral (e.g., intravenous, subcutaneous,intramuscular), intranasal, buccal, topical, rectal, or transdermaladministration routes. The pharmaceutical formulations described hereininclude, but are not limited to, aqueous liquid dispersions,self-emulsifying dispersions, solid solutions, liposomal dispersions,aerosols, solid dosage forms, powders, immediate release formulations,controlled release formulations, fast melt formulations, tablets,capsules, pills, delayed release formulations, extended releaseformulations, pulsatile release formulations, multiparticulateformulations, and mixed immediate and controlled release formulations.

Pharmaceutical compositions including a compound described herein areoptionally manufactured in a conventional manner, such as, by way ofexample only, by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orcompression processes.

In some embodiments, a pharmaceutical compositions described hereinincludes one or more agents described herein, as an active ingredient infree-acid or free-base form, or in a pharmaceutically acceptable saltform. In some embodiments, the compounds described herein are utilizedas an N-oxide or in a crystalline or amorphous form (i.e., a polymorph).In certain embodiments, an active metabolite or prodrug of a compounddescribed herein is utilized. In some situations, a compound describedherein exists as tautomers. All tautomers are included within the scopeof the compounds presented herein. In certain embodiments, a compounddescribed herein exists in an unsolvated or solvated form, whereinsolvated forms comprise any pharmaceutically acceptable solvent, e.g.,water, ethanol, and the like. The solvated forms of the compoundspresented herein are also considered to be disclosed herein.

A “carrier” includes, in some embodiments, a pharmaceutically acceptableexcipient and is selected on the basis of compatibility with compoundsdisclosed herein, such as, compounds of any of Formulas I-V, and therelease profile properties of the desired dosage form. Exemplary carriermaterials include, e.g., binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, diluents, and the like. See, e.g.,Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

Moreover, in some embodiments, the pharmaceutical compositions describedherein are formulated as a dosage form. As such, in some embodiments,provided herein is a dosage form comprising a compound described herein,e.g., a compound of any of Formulas I-V, suitable for administration toan individual. In certain embodiments, suitable dosage forms include, byway of non-limiting example, aqueous oral dispersions, liquids, gels,syrups, elixirs, slurries, suspensions, solid oral dosage forms,aerosols, controlled release formulations, fast melt formulations,effervescent formulations, lyophilized formulations, tablets, powders,pills, dragees, capsules, delayed release formulations, extended releaseformulations, pulsatile release formulations, multiparticulateformulations, and mixed immediate release and controlled releaseformulations.

The pharmaceutical solid dosage forms described herein optionallyinclude an additional therapeutic compound described herein and one ormore pharmaceutically acceptable additives such as a compatible carrier,binder, filling agent, suspending agent, flavoring agent, sweeteningagent, disintegrating agent, dispersing agent, surfactant, lubricant,colorant, diluent, solubilizer, moistening agent, plasticizer,stabilizer, penetration enhancer, wetting agent, anti-foaming agent,antioxidant, preservative, or one or more combination thereof. In someaspects, using standard coating procedures, such as those described inRemington's Pharmaceutical Sciences, 20th Edition (2000), a film coatingis provided around the formulation of the compound of any of FormulaI-V. In one embodiment, a compound described herein is in the form of aparticle and some or all of the particles of the compound are coated. Incertain embodiments, some or all of the particles of a compounddescribed herein are microencapsulated. In some embodiment, theparticles of the compound described herein are not microencapsulated andare uncoated.

In some embodiments, the pharmaceutical composition described herein isin unit dosage forms suitable for single administration of precisedosages. In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of one or more compound. In someembodiments, the unit dosage is in the form of a package containingdiscrete quantities of the formulation. Non-limiting examples arepackaged tablets or capsules, and powders in vials or ampoules. Aqueoussuspension compositions are optionally packaged in single-dosenon-reclosable containers. In some embodiments, multiple-dosere-closeable containers are used. In certain instances, multiple dosecontainers comprise a preservative in the composition. By way of exampleonly, formulations for parenteral injection are presented in unit dosageform, which include, but are not limited to ampoules, or in multi-dosecontainers, with an added preservative.

In some embodiments, the agents and compositions described herein areadministered before, during, or after administration of a vaccine. Insome embodiments, the agents and compositions described herein areincorporated into a vaccine. In some embodiments, the agents andcompositions stimulate the immune system and increase the response ofthe immune system to a vaccine. In some embodiments, the agents andcompositions augment an immune response. In some embodiments, the agentsand compositions act synergistically with the vaccine. In someembodiments, the agents and compositions are vaccine adjuvants. In someembodiments, the vaccine formulation comprises an antigen or antigenicmoiety (e.g. a protein or polysaccharide from B. pertussis, C. tetani,E. coli, C. diphtheriae, P. Aeruginosa, V. cholerae, H. influenzae, N.meningitidis, S. pneumoniae, N. gonorrhea) and the agent. In someembodiments, the vaccine further comprises a carrier (e.g. water,saline, PBS), a preservative (e.g. thimerosal, 2-phenoxy ethanol,phenol, benzethonium chloride), a stabilizer (e.g. lactose, monosodiumglutamate), an antibiotic, an antioxidant, a pH controlling agent, orcombinations thereof.

Topical Formulations

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is administered topically (i.e., the polypeptide isadministered to the surface of the skin). In some embodiments, a MYCfusion polypeptide disclosed herein (e.g., TAT-MYC) is administeredtopically at the site of a vaccine injection. In certain instances, alocalized antigen depot (i.e., a tissue depot formed by vaccineadjuvants (e.g., water-in-oil emulsions, or aluminum salts) that resultsin the slow and even release of an antigen) arises during vaccinationwith current approaches. In some embodiments, the transient upregulationof MYC in the resident lymphoid cells in the skin, near the site of theantigen depot, drives a more robust immune response, and yields abroader response in the context of relaxed tolerance to self antigens.

Any suitable formulation is utilized. In some embodiments, the MYCfusion polypeptide is formulated as a solution, a cream, a lotion, agel, an ointment, a foam, a microemulsion, or a combination thereof. Insome embodiments, the MYC fusion polypeptide is formulated for deliveryvia a transdermal patch.

Any suitable method of topical administration is utilized (e.g.,electroporation, sonophoresis, chemical delivery, skin abrasion). Insome embodiments, the MYC fusion polypeptide is formulated for chemicaldelivery (i.e., a chemical substance (e.g., PEG, ethanol, glycerolmonolaurate, sodium dodecyl sulfate, phosphatidyl choline, or urea) isused to facilitate penetration of an active agent across the skin). Insome embodiments, the MYC fusion polypeptide is formulated for deliveryvia electroporation (i.e., the permeablization of a barrier (e.g., theskin) via application of an electric current). In some embodiments, theMYC fusion polypeptide is formulated for delivery via sonophoresis(i.e., the permeablization of a barrier (e.g., the skin) via applicationof ultrasound).

Creams and Lotions

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated as a cream. In certain instances, creams aresemisolid (e.g., soft solid or thick liquid) formulations that include aMYC fusion polypeptide disclosed herein (e.g., TAT-MYC) dispersed in anoil-in-water emulsion or a water-in-oil emulsion.

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated as a lotion. In certain instances, lotions arefluid emulsions (e.g., oil-in-water emulsions or a water-in-oilemulsions).

In some embodiments, the hydrophobic component of a lotion and/or creamis derived from an animal (e.g., lanolin, cod liver oil, and ambergris),plant (e.g., safflower oil, castor oil, coconut oil, cottonseed oil,menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil,rapeseed oil, linseed oil, rice bran oil, pine oil, sesame oil, orsunflower seed oil), petroleum (e.g., mineral oil, or petroleum jelly),or a combination thereof.

Ointments

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated as an ointment. In certain instances, ointmentsare semisolid preparations that soften or melt at body temperature.

Pastes

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated as a paste. In certain instances, pastes containat least 20% solids. In certain instances, pastes are ointments that donot flow at body temperature.

Gels

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated as a gel. In certain instances, gels aresemisolid (or semi-rigid) systems consisting of dispersions of largeorganic molecules dispersed in a liquid. In certain instances, gels arewater-soluble and are removed using warm water or saline.

Sticks

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated as a stick. In certain instances, sticks aresolid dosage forms that melt at body temperature. In some embodiments, astick comprises a wax, a polymer, a resin, dry solids fused into a firmmass, and/or fused crystals. In some embodiments, a topical formulationof a MYC fusion polypeptide disclosed herein (e.g., TAT-MYC) is in theform of a styptic pencil (i.e., a stick prepared by (1) heating crystalsuntil they lose their water of crystallization and become molten, and(2) pouring the molten crystals into molds and allowing them to harden).In some embodiments, a topical formulation of a MYC fusion polypeptidedisclosed herein (e.g., TAT-MYC) is in the form of stick wherein thestick comprises a wax (e.g., the wax is melted and poured intoappropriate molds in which they solidify in stick form).

In some embodiments, a topical formulation of a MYC fusion polypeptidedisclosed herein (e.g., TAT-MYC) is in the form of stick wherein thestick comprises a melting base (i.e., a base that softens at bodytemperature). Examples of melting bases include, but are not limited to,waxes, oils, polymers and gels. In some embodiments, a topicalformulation of a MYC fusion polypeptide disclosed herein (e.g., TAT-MYC)is in the form of stick wherein the stick comprises a moisten base(i.e., a base that is activated by the addition of moisture).

Patches

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated for administration via a patch. In someembodiments, a topical formulation disclosed herein is dissolved and/ordispersed in a polymer or an adhesive. In some embodiments, a patchdisclosed herein is constructed for continuous, pulsatile, or on demanddelivery of a MYC fusion polypeptide disclosed herein (e.g., TAT-MYC).

Wound Dressings

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated for administration via a wound dressing. Wounddressings include, but are not limited to gauzes, transparent filmdressings, hydrogels, polyurethane foam dressings, hydrocolloids andalginates.

Dermatological Excipients

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated with a penetration enhancer. Penetrationenhancers include, but are not limited to, sodium lauryl sulfate, sodiumlaurate, polyoxyethylene-20-cetyl ether, laureth-9, sodiumdodecylsulfate, dioctyl sodium sulfosuccinate, polyoxyethylene-9-laurylether (PLE), Tween 80, nonylphenoxypolyethylene (NP-POE), polysorbates,sodium glycocholate, sodium deoxycholate, sodium taurocholate, sodiumtaurodihydrofusidate, sodium glycodihydrofusidate, oleic acid, caprylicacid, mono- and di-glycerides, lauric acids, acylcholines, caprylicacids, acylcarnitines, sodium caprates, EDTA, citric acid, salicylates,DMSO, decylmethyl sulfoxide, ethanol, isopropanol, propylene glycol,polyethylene glycol, glycerol, propanediol, and diethylene glycolmonoethyl ether.

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated with a gelling (or thickening) agent. In someembodiments, a topical formulation disclosed herein further comprisesfrom about 0.1% to about 5%, more preferably from about 0.1% to about3%, and most preferably from about 0.25% to about 2%, of a gellingagent. In certain embodiments, the viscosity of a topical formulationdisclosed herein is in the range from about 100 to about 500,000 cP,about 100 cP to about 1,000 cP, about 500 cP to about 1500 cP, about1000 cP to about 3000 cP, about 2000 cP to about 8,000 cP, about 4,000cP to about 10,000 cP, about 10,000 cP to about 50,000 cP.

Suitable gelling agents for use in preparation of the gel topicalformulation include, but are not limited to, celluloses, cellulosederivatives, cellulose ethers (e.g., carboxymethylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose),guar gum, xanthan gum, locust bean gum, alginates (e.g., alginic acid),silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan,paraffin, petrolatum, acacia (gum arabic), agar, aluminum magnesiumsilicate, sodium alginate, sodium stearate, bladderwrack, bentonite,carbomer, carrageenan, carbopol, xanthan, cellulose, microcrystallinecellulose (MCC), ceratonia, chondrus, dextrose, furcellaran, gelatin,ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin,mannitol, sorbitol, honey, maize starch, wheat starch, rice starch,potato starch, gelatin, sterculia gum, polyethylene glycol (e.g. PEG200-4500), gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose,ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose,hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethylmethacrylate), oxypolygelatin, pectin, polygeline, povidone, propylenecarbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA),poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate),hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodiumcarboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone(PVP: povidone), or combinations thereof

In some embodiments, a MYC fusion polypeptide disclosed herein (e.g.,TAT-MYC) is formulated with an emollient. Emollients include, but arenot limited to, castor oil esters, cocoa butter esters, safflower oilesters, cottonseed oil esters, corn oil esters, olive oil esters, codliver oil esters, almond oil esters, avocado oil esters, palm oilesters, sesame oil esters, squalene esters, kikui oil esters, soybeanoil esters, acetylated monoglycerides, ethoxylated glycerylmonostearate, hexyl laurate, isohexyl laurate, isohexyl palmitate,isopropyl palmitate, methyl palmitate, decyloleate, isodecyl oleate,hexadecyl stearate decyl stearate, isopropyl isostearate, methylisostearate, diisopropyl adipate, diisohexyl adipate, dihexyldecyladipate, diisopropyl sebacate, lauryl lactate, myristyl lactate, andcetyl lactate, oleyl myristate, oleyl stearate, and oleyl oleate,pelargonic acid, lauric acid, myristic acid, palmitic acid, stearicacid, isostearic acid, hydroxystearic acid, oleic acid, linoleic acid,ricinoleic acid, arachidic acid, behenic acid, erucic acid, laurylalcohol, myristyl alcohol, cetyl alcohol, hexadecyl alcohol, stearylalcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol,ricinoleyl alcohol, behenyl alcohol, erucyl alcohol, 2-octyl dodecanylalcohol, lanolin and lanolin derivatives, beeswax, spermaceti, myristylmyristate, stearyl stearate, carnauba wax, candelilla wax, lecithin, andcholesterol.

Combinations

In some embodiments, it is appropriate to administer at least onetherapeutic agent described herein in combination with anothertherapeutic agent. Or, by way of example only, the benefit experiencedby a patient is increased by administering one of the compoundsdescribed herein with another therapeutic agent (which also includes atherapeutic regimen) that also has therapeutic benefit. In any case,regardless of the disorder, disease or condition being treated, theoverall benefit experienced by the patient is, in some embodiments,additive of the two therapeutic agents or in other embodiments, thepatient experiences a synergistic benefit.

In some embodiments, the particular choice of compounds depends upon thediagnosis of the attending physicians and their judgment of thecondition of the patient and the appropriate treatment protocol. Thecompounds are optionally administered concurrently (e.g.,simultaneously, essentially simultaneously or within the same treatmentprotocol) or sequentially, depending upon the nature of the disease,disorder, or condition, the condition of the patient, and the actualchoice of compounds used. In certain instances, the determination of theorder of administration, and the number of repetitions of administrationof each therapeutic agent during a treatment protocol, is based on anevaluation of the disorder being treated and the condition of thepatient.

In some embodiments, therapeutically-effective dosages vary when thedrugs are used in treatment combinations. Methods for experimentallydetermining therapeutically-effective dosages of drugs and other agentsfor use in combination treatment regimens are described in theliterature. For example, the use of metronomic dosing, i.e., providingmore frequent, lower doses in order to minimize toxic side effects, hasbeen described extensively in the literature. Combination treatmentfurther includes periodic treatments that start and stop at varioustimes to assist with the clinical management of the patient.

In some embodiments of the combination therapies described herein,dosages of the co-administered compounds vary depending on the type ofco-drug employed, on the specific drug employed, on the disorder orcondition being treated and so forth. In addition, when co-administeredwith one or more biologically agents, the compound provided herein isoptionally administered either simultaneously with the biologicallyagent(s), or sequentially. In certain instances, if administeredsequentially, the attending physician will decide on the appropriatesequence of therapeutic compound described herein in combination withthe additional therapeutic agent.

The multiple therapeutic agents (at least one of which is a therapeuticcompound described herein) are optionally administered in any order oreven simultaneously. If simultaneously, the multiple therapeutic agentsare optionally provided in a single, unified form, or in multiple forms(by way of example only, either as a single pill or as two separatepills). In certain instances, one of the therapeutic agents isoptionally given in multiple doses. In other instances, both areoptionally given as multiple doses. If not simultaneous, the timingbetween the multiple doses is any suitable timing, e.g., from more thanzero weeks to less than four weeks. In some embodiments, the additionaltherapeutic agent is utilized to achieve remission (partial or complete)of a cancer, whereupon the therapeutic agent described herein (e.g., acompound of any one of Formulas I-V) is subsequently administered. Inaddition, the combination methods, compositions and formulations are notto be limited to the use of only two agents; the use of multipletherapeutic combinations are also envisioned (including two or moretherapeutic compounds described herein).

In some embodiments, a dosage regimen to treat, prevent, or amelioratethe condition(s) for which relief is sought, is modified in accordancewith a variety of factors. These factors include the disorder from whichthe subject suffers, as well as the age, weight, sex, diet, and medicalcondition of the subject. Thus, in various embodiments, the dosageregimen actually employed varies and deviates from the dosage regimensset forth herein.

In some embodiments, the pharmaceutical agents which make up thecombination therapy disclosed herein are provided in a combined dosageform or in separate dosage forms for substantially simultaneousadministration. In certain embodiments, the pharmaceutical agents thatmake up the combination therapy are administered sequentially, witheither therapeutic compound being administered by a regimen calling fortwo-step administration. In some embodiments, two-step administrationregimen calls for sequential administration of the agents orspaced-apart administration of the separate agents. In certainembodiments, the time period between the multiple administration stepsvaries, by way of non-limiting example, from a few minutes to severalhours, depending upon the properties of each pharmaceutical agent, suchas potency, solubility, bioavailability, plasma half-life and kineticprofile of the pharmaceutical agent.

In addition, the compounds described herein also are optionally used incombination with procedures that provide additional or synergisticbenefit to the patient. By way of example only, patients are expected tofind therapeutic and/or prophylactic benefit in the methods describedherein, wherein pharmaceutical composition of a compound disclosedherein and /or combinations with other therapeutics are combined withgenetic testing to determine whether that individual is a carrier of agene or gene mutation that is known to be correlated with certaindisorders or conditions. In certain embodiments, prophylactic benefit isachieved by administering a therapeutic compound described herein to anindividual whose proliferative disorder (e.g., cancer) is in remission(e.g., partial or complete).

In some embodiments, the compounds described herein and combinationtherapies are administered before, during or after the occurrence of adisorder or condition. Timing of administering the compositioncontaining a compound is optionally varied to suit the needs of theindividual treated. Thus, in certain embodiments, the compounds are usedas a prophylactic and are administered continuously to subjects with apropensity to develop conditions or disorders in order to prevent theoccurrence of the disorder or condition. In some embodiments, thecompounds and compositions are administered to an individual during oras soon as possible after the onset of the symptoms. The administrationof the compounds is optionally initiated within the first 48 hours ofthe onset of the symptoms, within the first 6 hours of the onset of thesymptoms, or within 3 hours of the onset of the symptoms. The initialadministration is achieved by any route practical, such as, for example,an intravenous injection, a bolus injection, infusion over 5 minutes toabout 5 hours, a pill, a capsule, transdermal patch, buccal delivery,and the like, or combination thereof. In some embodiments, the compoundshould be administered as soon as is practicable after the onset of adisorder or condition is detected or suspected, and for a length of timenecessary for the treatment of the disorder, such as, for example, frommore than 1 month to about 3 months. The length of treatment isoptionally varied for each subject based on known criteria. In exemplaryembodiments, the compound or a formulation containing the compound isadministered for at least 2 weeks, between more than 1 month to about 5years, or from more than 1 month to about 3 years.

In some embodiments, therapeutic agents are combined with or utilized incombination with one or more of the following therapeutic agents in anycombination: Akt selective inhibitors; PI3K inhibitors; polypeptidekinase C (PKC) inhibitors; farnesyltransferase inhibitors; inhibitors ofsarco/endoplasmic reticulum Ca2+ ATPase; Ca⁺⁺/calmodulin (CaM)-dependentpolypeptide kinase inhibitors; cyclin-dependent kinase inhibitors; PPD(p-phenylenediamine); D609 (tricyclodecan-9-yl xanthogenate); PP1(4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine);AG-879 (alpha-Cyano-(3,5-di-t-butyl-4-hydroxy)thiocinnamide); BAY11-7082 ((E)-3-(4-Methylphenylsulfonyl)-2-propenenitrile); thapsigargin,or RK-682 (3-Hexadecanoyl-5-hydroxymethyl-tetronic acid).

Immunosuppressants include, by way of non-limiting example, Sirolimus(rapamycin), cyclosporin, FK506, cypermethrin, deltamethrin,fenvalerate, tyrphostin 8, methotrexate, piceatannol, genistein,tacrolimus, rapamicin, cyclophosphamide, azathioprine, mercaptopurine,mycophenolate, and FTY720.

Akt selective inhibitors include, by way of non-limiting example, SH4(1L-6-hydroxymethyl-chiro-inositol2-R-2-O-methyl-3-O-octadecylcarbonate); SH-5 (D-3-deoxy-2-O-methyl-myoinositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate);and SH-6 (D-2,3-dieoxy-myo inositol 1-(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate).

PI3K inhibitors include, by way of non-limiting example, wortmannin;LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one); D000(Upstate, Milton Keynes, United Kingdom); and D121(3-phenyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one).

By way of non-limiting example, protein kinase C (PKC) inhibitorsinclude Ro-318220(3-[1-[3-(amidinothio)propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl) maleimide methane sulfonate); Gö6976(12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole);staurosporin; rottlerin; calphastin C; GF 109203X(3-[1-(Dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimidehydrochloride); hypericin; sphingosine; miltefosine;palmitoyl-DL-carnitine Cl; rottlerin; and2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimenthol dimethylether.

By way of non-limiting example, farmesyltransferase inhibitors includeL-744,832 (C₂₆H₄₅N₃O₆S₂ 2HCl); or Sarasar (SCH66336 or lonafarnib).

By way of non-limiting example, Ca⁺⁺/calmodulin (CaM)-dependentpolypeptide kinase inhibitors include:2-hydroxy-5-(2,5-dihydroxybenzylamino) benzoic acid; KN-62(1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine);KN-93 (2[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine);or staurosporine (AM-2282).

By way of non-limiting example, cyclin-dependent kinase inhibitorsinclude: roscovitine (Seliciclib or CYC202); purvalanol A; or indirubin.

Methods of Dosing and Treatment Regimens

In some embodiments, an agent and/or composition described herein isused in the preparation of vaccines for immunization against pathogens(e.g. hepatitis A; hepatitis B; polio; measles; mumps; rubella;diphtheria; pertussis; tetanus; influenza; varicella zoster virus;rotavirus; influenza; meningococcal disease; pneumonia; smallpox;cholera; bubonic plague; yellow fever; tuberculosis: human paplomavirus)wherein an up-regulation of an immune response would be beneficial tosaid treatment. In some embodiments, an agent described herein isincorporated into the formulation of a vaccine. In some embodiments, anagent described herein is a vaccine adjuvant. In some embodiments, thevolume of a vaccine formulation for injection is from about 50 μl toabout 5 ml. In some embodiments, the volume of a vaccine formulation forinjection is from about 100 μl to about 3.5ml. In some embodiments, thevolume of a vaccine formulation for injection is from about 200 μl toabout 2 ml. In some embodiments, the volume of a vaccine formulation forinjection is from about 350 μl to about 1 ml. In some embodiments, thevolume of a vaccine formulation for injection is about 500 μl. Thevolume of the vaccine is determined by the method of administration. Incertain instances, the vaccine is administered as a subcutaneous orintradermal injection. In certain instances, the vaccine is administeredas intramuscular injection. In some embodiments, the vaccine isadministered as 1 dose. In some embodiments, the vaccine is administeredin multiple doses (e.g. in booster shots).

In some embodiments, an agent and/or composition described herein isused in the preparation of medicaments for the prophylactic and/ortherapeutic treatment of disorders that would benefit, at least in part,from a down-regulation of an immune response (e.g. autoimmune disorders,transplant rejection).

In some embodiments, an agent and/or composition described herein isused in a method of treating a disorder (e.g. hepatitis A; hepatitis B;polio; measles; mumps; rubella; diphtheria; pertussis; tetanus;influenza; varicella zoster virus; rotavirus; influenza; meningococcaldisease; pneumonia; smallpox; cholera; bubonic plague; yellow fever;tuberculosis: human paplomavirus, and a cancer) wherein an up-regulationof an immune response would be beneficial to said treatment. In someembodiments, an agent and/or composition described herein is used in amethod of prophylactic and/or therapeutic treatment of a disorder thatwould benefit, at least in part, from a down-regulation of an immuneresponse (e.g. autoimmune disorders, transplant rejection).

In certain instances wherein the patient's condition does not improve,upon the doctor's discretion the administration of an agent orcomposition described herein is optionally administered chronically,that is, for an extended period of time, including throughout theduration of the patient's life in order to ameliorate or otherwisecontrol or limit the symptoms of the patient's disorder.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of an agent or composition describedherein is optionally given continuously; alternatively, the dose of drugbeing administered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of thesymptoms, to a level at which the improved disease, disorder orcondition is retained. In some embodiments, patients requireintermittent treatment on a long-term basis upon any recurrence ofsymptoms.

In some embodiments, the agents or compositions described herein are inunit dosage forms suitable for single administration of precise dosages.In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of an agent or composition describedherein. In some embodiments, the unit dosage is in the form of a packagecontaining discrete quantities of the formulation. Non-limiting examplesare packaged tablets or capsules, and powders in vials or ampoules. Insome embodiments, aqueous suspension compositions are packaged insingle-dose non-reclosable containers. Alternatively, multiple-dosereclosable containers are used, in which case it is typical to include apreservative in the composition. By way of example only, formulationsfor parenteral injection are presented in unit dosage form, whichinclude, but are not limited to ampoules, or in multi dose containers,with an added preservative.

The daily dosages appropriate for an agent or composition describedherein are from about 0.01 to 10.0 mg/kg per body weight. In someembodiments, the daily dosages appropriate for an agent or compositiondescribed herein are from about 0.05 to 7.5 mg/kg per body weight. Insome embodiments, the daily dosages appropriate for an agent orcomposition described herein are from about 0.1 to 5.0 mg/kg per bodyweight. In some embodiments, the daily dosages appropriate for an agentor composition described herein are from about 0.25 to 2.5 mg/kg perbody weight. In some embodiments, the daily dosages appropriate for anagent or composition described herein are from about 0.5 to 1.0 mg/kgper body weight. An indicated daily dosage in the larger mammal,including, but not limited to, humans, is in the range from about 0.5 mgto about 100 mg, conveniently administered in divided doses, including,but not limited to, up to four times a day or in extended release form.Suitable unit dosage forms for oral administration include from morethan 1 to 50 mg active ingredient. The foregoing ranges are merelysuggestive, as the number of variables in regard to an individualtreatment regime is large, and considerable excursions from theserecommended values are not uncommon. Such dosages are optionally altereddepending on a number of variables, not limited to the activity of theagent or composition described herein used, the disorder or condition tobe treated, the mode of administration, the requirements of theindividual subject, the severity of the disorder or condition beingtreated, and the judgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD50 (the doselethal to 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD50 and EDS50. An agent or compositions describedherein exhibiting high therapeutic indices is preferred. The dataobtained from cell culture assays and animal studies are optionally usedin formulating a range of dosage for use in human. The dosage of such anagent or composition described herein lies preferably within a range ofcirculating concentrations that include the ED50 with minimal toxicity.The dosage optionally varies within this range depending upon the dosageform employed and the route of administration utilized.

EXAMPLES

The following examples are for illustrative purposes only and arenon-limiting embodiments. Many modifications, equivalents, andvariations of the present invention are possible in light of the aboveteachings, therefore, it is to be understood that within the scope ofthe appended claims, the invention may be practiced other than asspecifically described.

Example 1 Assay Using a Reporter Construct

A plurality of cells are transformed with a reporter construct. Thereporter construct has FLuc under control of an ODC promoter. The cellsare divided into 96 aliquots using a standard 96-well plate. Each wellis administered a different small molecule. The small molecule isincubated with the cells for 6 hours. The level of expression of theluciferase is determined using fluorescent microscopy. Agents whichinduce the expression of the luciferase gene are validated in vitro.

Example 2 Construction of a TAT-MYC Fusion Peptide

Construction of p-TAT-MYC

Plasmid pTAT-Myc-V5-6xHis was made by PCR amplification of the codingregions for human MYC using a forward primer that contains an in frameN-terminal 9-amino-acid sequence of the TAT protein transduction domainof HIV-1 (RKKRRQRRR), and a reverse primer that removed the stop codon.The PCR product was then cloned into pET101/D-Topo (Invitrogen) vector,which includes a C-terminal V5 epitope and 6×-histidine purificationtag.

Bacterial Strain Used for Protein Expression

BL-21 RARE cells were created by transforming BL-21 Star™ E. coli strain(Invitrogen) with pRARE (CamR), isolated from BL21 Rosetta cells(Novagen), that express tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons.

Protein Induction and Purification

pTAT-Myc-V5-6×His was transformed into BL21 RARE cells and grown onTB/Amp/Cam plate at 37° C. overnight. An isolated colony was used toinoculate a 5 ml TB/Amp/Cam starter culture and grown at 37° C.overnight. 1 liter of TB/Amp/Cam broth was inoculated with the 5 mlstarter culture and grown to an OD600 of 0.5, and induced with 0.5mMIPTG at 37° C. for 3 hrs.

Bacterial cells were pelleted by centrifugation and the cell pellet wasresuspended in lysis buffer (8 M urea, 100 mM NaH₂PO₄, 10 mM Tris pH to8.0) and lysed at room temperature overnight on a shaker. The lysate wascleared by centrifugation at 29,000×g for 30 min and the supernatant wasapplied to a nickel nitrilotriacetate affinity column (Qiagen) usinggravity flow. The column was washed with 25 volumes of lysis buffercontaining 10 mM imidazole followed by elution with lysis buffercontaining 100 mM imidazole.

Protein was concentrated using an Amicon Ultra centrifugal filter device(10,000 MWCO) and dialyzed in a stepwise fashion into dialysis buffer(50 mM NaH₂PO₄, 5 mM Tris pH 7.0, 450 mM NaCl, 5% glycerol, 1 mM DTT).The dialysis went as follows: 2 hours in dialysis buffer containing 4Murea, 2 hours in buffer with 2M urea, then 2 hrs in dialysis bufferalone. Purity and size of proteins were verified using SDS-PAGEelectrophoresis and either coomassie blue staining or western blot withanti-V5 (1:5000; Invitrogen) or anti-c-Myc (N-262, 1:2000; Santa CruzBiotechnology) antibodies.

Protein concentration was measured by Bradford protein assay (Sigma)compared to a standard curve of bovine serum albumin.

Example 3 Effect of TAT-Myc Fusion Peptide on Immune Responses

The TAT-MYC fusion peptide is evaluated in vitro as a regulator of animmune response. A primary T-cell activation culture is incubated withthe TAT-MYC fusion peptide for 72 hours. The culture is then stainedwith CFSE. Following staining with CFSE, the culture is analyzed byFACS.

Example 4 Effect of TAT-Myc Fusion Peptide on Immune Responses

The TAT-MYC fusion peptide is evaluated in vivo as a regulator of animmune response. A mouse is immunized against NP-KHL by administeringNP-KHL to the mouse. Immediately after immunization against the antigen,the TAT-MYC fusion peptide is administered to the mouse. Twenty-fourhours after the immunization serum is taken from the mouse. The serum isanalyzed for antibodies. Alternatively, the TAT-MYC fusion peptide isadministered concurrently with immunization, or preceding immunization.

Example 5 Effect of IM TAT-MYC as a Vaccine Adjuvant

This will be a double-blind, randomized, placebo-controlled study. It isdesigned to assess immunological memory following administration of aninfluenza vaccine and a TAT-MYC fusion peptide.

Participants are divided into two groups. Group I will be administeredan influenza vaccine and saline. Group II will be administered aninfluenza vaccine and the TAT-MYC fusion peptide.

Primary Objective

To assess the immunological effects of a TAT-MYC fusion peptideadministered by intramuscular (IM) injection to individuals receiving aninfluenza vaccine.

Secondary Objectives

To assess the safety and tolerability of a TAT-MYC fusion peptideadministered by intramuscular injection to individuals receiving aninfluenza vaccine.

Methodology

On Day=0, both Group I and Group II are administered a single dose ofthe same influenza vaccine. Subjects are monitored for 2 hours fornegative side-effects.

All individuals in Group I not exhibiting negative side-effects areadministered 20 uL of saline by IM injection.

All individuals in Group II not exhibiting negative side-effects areadministered 20 uL of the TAT-MYC fusion peptide by IM injection.

Blood is drawn from all individuals on Day=1. T-cell counts aredetermined and recorded.

On Day=20, all individuals are challenged with antigens from relevantinfluenza strains. Blood is drawn for all individuals 1 hour afterchallenge, 6 hours after challenge, 12 hours after challenge, and 24hours after challenge. T-cell levels are determined.

Example 6 Effect of Topical TAT-MYC as a Vaccine Adjuvant

This will be a double-blind, randomized, placebo-controlled study. It isdesigned to assess immunological memory following administration of aninfluenza vaccine and a TAT-MYC fusion peptide.

Participants are divided into two groups. Group I will be administeredan influenza vaccine and saline. Group II will be administered aninfluenza vaccine and the TAT-MYC fusion peptide.

Primary Objective

To assess the immunological effects of a TAT-MYC fusion peptideadministered topically to individuals receiving an influenza vaccine.

Secondary Objectives

To assess the safety and tolerability of a TAT-MYC fusion peptideadministered topically to individuals receiving an influenza vaccine.

Methodology

On Day=0, both Group I and Group II are administered a single dose ofthe same influenza vaccine. Subjects are monitored for 2 hours fornegative side-effects.

All individuals in Group I not exhibiting negative side-effects aregiven one application of the placebo lotion. Individuals are instructedto apply the lotion onto the skin at the site of the vaccination.

All individuals in Group II not exhibiting negative side-effects aregiven one application of the TAT-MYC lotion. Individuals are instructedto apply the lotion onto the skin at the site of the vaccination.

Blood is drawn from all individuals on Day=1. T-cell counts aredetermined and recorded.

On Day=20, all individuals are challenged with antigens from relevantinfluenza strains. Blood is drawn for all individuals 1 hour afterchallenge, 6 hours after challenge, 12 hours after challenge, and 24hours after challenge. T-cell levels are determined.

Example 7 Effect on RA of an Agent that Down-Regulates MYC ExpressionPrimary Objective

To assess the steady-state trough serum concentrations of an agent thatdecreases the expression of MYC as identified by a method disclosedherein (hereinafter, “Agent X”) following weekly subcutaneous dosing insubjects with active rheumatoid arthritis (RA).

Secondary Objectives

To assess the safety and tolerability of Agent X administeredsubcutaneously to individuals with RA; to assess the immunogenicity ofAgent X administered subcutaneously in individuals with RA; and toexamine the effect of subcutaneous administration of Agent X on serumlevels of rheumatoid factor (RF) in individuals with RA.

Methodology

This will be a double-blind, randomized, placebo-controlled,parallel-group, multiple-dose study. It will be designed to assess thesteady-state trough serum concentrations of Agent X followingsubcutaneous administration in subjects with RA.

Subjects will be randomized according to a computer generatedrandomization scheme, in a 3:1 ratio, to receive either Agent X orplacebo in 1 of 5 parallel groups based on body weight obtained at ascreening visit.

On Day 1, subjects will receive a single IV infusion (loading dose) ofAgent X or placebo, based on their weight range. Agent X or placebo willbe administered intravenously over ˜30 minutes using a calibrated,constant-rate infusion pump prior to starting subcutaneous treatment.Approximately 1 hour after the completion of the IV infusion, subjectswill receive their assigned subcutaneous dose of Agent X or placebodrawn from a vial or using a pre-filled syringe. Agent X or placebo willbe administered by clinical study site staff weekly by the subcutaneousroute, at the same dose as the subcutaneous dose on Day 1 for a total of12 subcutaneous injections.

Subjects will be monitored for adverse events (AEs) throughout theduration of the study. Physical examinations, vital sign measurements,and clinical laboratory evaluations will be performed at selected timesthroughout the study. Blood samples for PK analysis will be collected onDay 1 prior to and at the end of the IV infusion. In addition, bloodsamples will be collected prior to each weekly SC dose of agent X onDays 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 78 for the assessment ofsteady-state Cmin concentrations. A single blood sample will also becollected on Days 72, 74, 75, and 76 for evaluation of steady-stateCmax, Tmax, and AUC(TAU), where TAU=7 days, and at study discharge (Day85). Blood samples for assessment of immunogenicity will be obtainedprior to the administration of agent X on Days 1, 15, 29, 43, 57, 71,and 85. Blood samples for the determination of RF will be obtained priorto dosing on Days 1, 8, 15, 29, 57, and 85.

Subjects who complete the 12 weeks of subcutaneous dosing of Agent X orplacebo are eligible to enter into a long term extension (LTE). Forsubjects who entered the LTE, the first dose of Agent X will beadministered on Day 85.

Number of Subjects

Between 48 and 72 subjects are to be randomized to study treatment.

Diagnosis and Criteria for Inclusion

Men or women with active RA who are above 18 will eligible forparticipation in this study. Subjects must meet the classificationcriteria of the American College of Rheumatology (ACR) (formerlyAmerican Rheumatism Association (ARA)), for RA and had active disorder.Subjects must have had RA for at least 1 year from the time of initialdiagnosis.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-20. (canceled)
 21. A method, comprising: (a) providing a fusionpeptide to one or more immune cells in vitro, wherein the fusion peptidecomprises: (i) a transporter peptide sequence; and (ii) a MYCpolypeptide sequence, and wherein the one or immune cells have beenexposed to an antigen; and (b) culturing the one or more immune cellsunder conditions sufficient for the fusion peptide to increase one ormore of activation, survival, or proliferation of the one or more immunecells, as compared with corresponding immune cells not cultured with thefusion peptide.
 22. The method of claim 21, wherein increasingactivation comprises one or more of decreasing the amount of time ittakes for the one or more immune cells to respond to the antigen orincreasing the rate or amount the one or more immune cells proliferatein response to the antigen.
 23. The method of claim 21, whereinincreasing proliferation comprises increasing the rate or amount the oneor more immune cells proliferate.
 24. The method of claim 21, whereinincreasing survival comprises increasing the length of time the one ormore immune cells survive.
 25. The method of claim 21, wherein the oneor more immune cells comprise T cells.
 26. The method of claim 21,wherein the one or more immune cells comprise B cells.
 27. The method ofclaim 21, wherein the one or more immune cells are cultured in theabsence of a cytokine.
 28. The method of claim 21, wherein the one ormore immune cells are one or more activated immune cells.
 29. The methodof claim 21, wherein the fusion peptide has Formula (I): transporterpeptide sequence—MYC polypeptide sequence.
 30. he method of claim 21,wherein the fusion peptide has Formula (II): transporter peptidesequence—X—MYC polypeptide sequence, wherein —X— is the one or moremolecules that link the transporter peptide sequence and the MYCpolypeptide sequence.
 31. he method of claim 21, wherein the fusionpeptide has Formula (II): transporter peptide sequence—X—MYC sequence,wherein in —X— is at least one amino acid that links the transporterpeptide sequence and the MYC polypeptide sequence.
 32. The method ofclaim 21, wherein the antigen is a peptide, a nucleic acid sequence, ora polysaccharide; derived from a neoplastic cell, a pathogen, or atoxoid; or a combination thereof.
 33. The method of claim 21, whereinthe antigen is derived from a pathogen selected from the groupconsisting of hepatitis A; hepatitis B; polio; measles; mumps; rubella;diphtheria; pertussis; tetanus; influenza; varicella zoster virus;rotavirus; meningococcal; pneumonia; smallpox; cholera; bubonic plague;yellow fever; tuberculosis; human papillomavirus; and combinationsthereof.